Dissertations / Theses on the topic 'Porcine'

Two types of porcine circovirus (PCV) exist, referred to as PCV1 and PCV2. PCV2 has been associated with disease syndromes in pigs, including that designated postweaning multisystemic wasting syndrome (PMWS), which has been identified in all regions of the world bar Australia (Hamel et al., 1998; Allan et al., 1999a; Onuki et al., 1999; Martelli et al., 2000; Kyriakis et al., 2000; Wellenberg et al., 2000; Done et al., 2001; Trujano et al., 2001; Saradell et al., 2004; Castro et al., 2004; Jemersic et al., 2004; Maldonado et al., 2004; Wang et al., 2004; Motovski and Segales, 2004; Garkavenko et al., 2005). PMWS affects young weaner pigs and results in weight loss, tachypnea, dyspnea, enlarged lymph nodes and jaundice (Harding, 1998). PCV2 may also cause or contribute to other swine diseases such as congential tremors (CT) (Stevenson et al., 1999), porcine dermatitis and nephropathy syndrome (PDNS) (Rosell et al., 2000), reproductive failure (Meehan et al., 2001) and several other emerging disease syndromes. PCV1 is currently considered to be non-pathogenic. Although PMWS has not been reported in Australia, information on the distribution, variation and further characterisation of PCV in Australian pigs was necessary as it might provide insights into why there is no PCV-associated disease in this country. The results reported in this thesis involved the detection and further study of porcine circovirus in Australia. This chapter provides an outline of this thesis and the work undertaken, while Chapter 2 is a review of the relevant literature with particular reference to circoviral diseases. Chapter 3 describes the detection of both PCV1 and PCV2 in the Australian pig herd, using a multiplex PCR designed to differentiate between the two viral types. The association of Australian PCV with two disease outbreaks was also investigated. Following the detection of both viruses, it was important to genetically compare Australian PCV with overseas strains known to cause disease, and this was achieved with a sequencing and phylogenetic study as described in Chapter 4. Possible reasons for the genetic groupings and distribution of different PCV2 strains worldwide are also discussed in this chapter. As PMWS is as yet unidentified in Australian pigs, the importation of pig meat into Australia from countries with the disease requires careful monitoring. Current protocols for the cooking of imported pig meat were designed to inactivate porcine reproductive and respiratory disease virus (PRRSV), and as such may not be effective against PCV. In this study (Chapter 5), Australian PCV2 was successfully infected into cell culture, and detected using a variety of techniques. Subsequently, thermal stability experiments were performed using a newly-developed immunoperoxidase (IPMA) test. It was anticipated that this study would determine whether current importation protocols require revision, and the results would suggest that this is the case, with PCV2 unaffected by treatment comparable with current cooking protocols. While no animal experiments were undertaken in this study, it may become necessary to infect pigs with Australian PCV to determine viral pathogenicity. Cell culture inoculums have been used in the past overseas, but problems with contamination and viral titre have been encountered (Fenaux et al., 2001). Viral infectious clones can be used to overcome these problems, so an infectious clone of Australian PCV2 was constructed, as described in Chapter 6. While time constraints prevented the clone from being infected into culture, it is anticipated that the construct would be infectious as it is based on a previously published method (Hattermann et al., 2004). Chapter 7 is a general discussion of the results and conclusions from this study. The detection and characterisation of Australian PCV as described in this study has provided further information on the status of PCV in the Australian pig herd, and also developed diagnostic tests to assist in future research. These tools will be important when assessing and managing the risk of Australia experiencing PCV-associated diseases.

Two types of porcine circovirus (PCV) exist, referred to as PCV1 and PCV2. PCV2 has been associated with disease syndromes in pigs, including that designated postweaning multisystemic wasting syndrome (PMWS), which has been identified in all regions of the world bar Australia (Hamel et al., 1998; Allan et al., 1999a; Onuki et al., 1999; Martelli et al., 2000; Kyriakis et al., 2000; Wellenberg et al., 2000; Done et al., 2001; Trujano et al., 2001; Saradell et al., 2004; Castro et al., 2004; Jemersic et al., 2004; Maldonado et al., 2004; Wang et al., 2004; Motovski and Segales, 2004; Garkavenko et al., 2005). PMWS affects young weaner pigs and results in weight loss, tachypnea, dyspnea, enlarged lymph nodes and jaundice (Harding, 1998). PCV2 may also cause or contribute to other swine diseases such as congential tremors (CT) (Stevenson et al., 1999), porcine dermatitis and nephropathy syndrome (PDNS) (Rosell et al., 2000), reproductive failure (Meehan et al., 2001) and several other emerging disease syndromes. PCV1 is currently considered to be non-pathogenic. Although PMWS has not been reported in Australia, information on the distribution, variation and further characterisation of PCV in Australian pigs was necessary as it might provide insights into why there is no PCV-associated disease in this country. The results reported in this thesis involved the detection and further study of porcine circovirus in Australia. This chapter provides an outline of this thesis and the work undertaken, while Chapter 2 is a review of the relevant literature with particular reference to circoviral diseases. Chapter 3 describes the detection of both PCV1 and PCV2 in the Australian pig herd, using a multiplex PCR designed to differentiate between the two viral types. The association of Australian PCV with two disease outbreaks was also investigated. Following the detection of both viruses, it was important to genetically compare Australian PCV with overseas strains known to cause disease, and this was achieved with a sequencing and phylogenetic study as described in Chapter 4. Possible reasons for the genetic groupings and distribution of different PCV2 strains worldwide are also discussed in this chapter. As PMWS is as yet unidentified in Australian pigs, the importation of pig meat into Australia from countries with the disease requires careful monitoring. Current protocols for the cooking of imported pig meat were designed to inactivate porcine reproductive and respiratory disease virus (PRRSV), and as such may not be effective against PCV. In this study (Chapter 5), Australian PCV2 was successfully infected into cell culture, and detected using a variety of techniques. Subsequently, thermal stability experiments were performed using a newly-developed immunoperoxidase (IPMA) test. It was anticipated that this study would determine whether current importation protocols require revision, and the results would suggest that this is the case, with PCV2 unaffected by treatment comparable with current cooking protocols. While no animal experiments were undertaken in this study, it may become necessary to infect pigs with Australian PCV to determine viral pathogenicity. Cell culture inoculums have been used in the past overseas, but problems with contamination and viral titre have been encountered (Fenaux et al., 2001). Viral infectious clones can be used to overcome these problems, so an infectious clone of Australian PCV2 was constructed, as described in Chapter 6. While time constraints prevented the clone from being infected into culture, it is anticipated that the construct would be infectious as it is based on a previously published method (Hattermann et al., 2004). Chapter 7 is a general discussion of the results and conclusions from this study. The detection and characterisation of Australian PCV as described in this study has provided further information on the status of PCV in the Australian pig herd, and also developed diagnostic tests to assist in future research. These tools will be important when assessing and managing the risk of Australia experiencing PCV-associated diseases.

Two types of porcine circovirus (PCV) exist, referred to as PCV1 and PCV2. PCV2 has been associated with disease syndromes in pigs, including that designated postweaning multisystemic wasting syndrome (PMWS), which has been identified in all regions of the world bar Australia (Hamel et al., 1998; Allan et al., 1999a; Onuki et al., 1999; Martelli et al., 2000; Kyriakis et al., 2000; Wellenberg et al., 2000; Done et al., 2001; Trujano et al., 2001; Saradell et al., 2004; Castro et al., 2004; Jemersic et al., 2004; Maldonado et al., 2004; Wang et al., 2004; Motovski and Segales, 2004; Garkavenko et al., 2005). PMWS affects young weaner pigs and results in weight loss, tachypnea, dyspnea, enlarged lymph nodes and jaundice (Harding, 1998). PCV2 may also cause or contribute to other swine diseases such as congential tremors (CT) (Stevenson et al., 1999), porcine dermatitis and nephropathy syndrome (PDNS) (Rosell et al., 2000), reproductive failure (Meehan et al., 2001) and several other emerging disease syndromes. PCV1 is currently considered to be non-pathogenic. Although PMWS has not been reported in Australia, information on the distribution, variation and further characterisation of PCV in Australian pigs was necessary as it might provide insights into why there is no PCV-associated disease in this country. The results reported in this thesis involved the detection and further study of porcine circovirus in Australia. This chapter provides an outline of this thesis and the work undertaken, while Chapter 2 is a review of the relevant literature with particular reference to circoviral diseases. Chapter 3 describes the detection of both PCV1 and PCV2 in the Australian pig herd, using a multiplex PCR designed to differentiate between the two viral types. The association of Australian PCV with two disease outbreaks was also investigated. Following the detection of both viruses, it was important to genetically compare Australian PCV with overseas strains known to cause disease, and this was achieved with a sequencing and phylogenetic study as described in Chapter 4. Possible reasons for the genetic groupings and distribution of different PCV2 strains worldwide are also discussed in this chapter. As PMWS is as yet unidentified in Australian pigs, the importation of pig meat into Australia from countries with the disease requires careful monitoring. Current protocols for the cooking of imported pig meat were designed to inactivate porcine reproductive and respiratory disease virus (PRRSV), and as such may not be effective against PCV. In this study (Chapter 5), Australian PCV2 was successfully infected into cell culture, and detected using a variety of techniques. Subsequently, thermal stability experiments were performed using a newly-developed immunoperoxidase (IPMA) test. It was anticipated that this study would determine whether current importation protocols require revision, and the results would suggest that this is the case, with PCV2 unaffected by treatment comparable with current cooking protocols. While no animal experiments were undertaken in this study, it may become necessary to infect pigs with Australian PCV to determine viral pathogenicity. Cell culture inoculums have been used in the past overseas, but problems with contamination and viral titre have been encountered (Fenaux et al., 2001). Viral infectious clones can be used to overcome these problems, so an infectious clone of Australian PCV2 was constructed, as described in Chapter 6. While time constraints prevented the clone from being infected into culture, it is anticipated that the construct would be infectious as it is based on a previously published method (Hattermann et al., 2004). Chapter 7 is a general discussion of the results and conclusions from this study. The detection and characterisation of Australian PCV as described in this study has provided further information on the status of PCV in the Australian pig herd, and also developed diagnostic tests to assist in future research. These tools will be important when assessing and managing the risk of Australia experiencing PCV-associated diseases.

As lesões cutâneas e queimaduras são considerados os principais causadores de danos e perdas dos tecidos moles. Em casos severos de trauma, os processos naturais de regeneração são insuficientes no reparo dos danos, resultando em lesões cutâneas crônicas. A desvitalização de matrizes homólogas ou heterólogas é uma alternativa para a produção de matrizes dérmicas. A pele porcina é bastante similar à pele humana, podendo ser utilizada como matriz de colágeno na regeneração de tecido mole. Além disso, ela tem como constituinte principal o colágeno tipo I, e, assim, pode ser utilizada em queimaduras de segundo grau. Este trabalho teve como objetivo a preparação e caracterização de matrizes extracelulares de colágeno tipo I por meio de hidrólise alcalina e reticulação com glutaraldeído (GA). As matrizes de colágeno foram obtidas a partir da hidrólise alcalina de pele porcina, com posterior reticulação com GA, em diferentes concentrações (0-0,1%) e tempos de reação (15 e 45 min). As matrizes foram caracterizadas através de determinação do conteúdo de elastina, estabilidade biológica (tripsina), Calorimetria Exploratória Diferencial (DSC), termogravimetria (TG/DTG), Microscopia Eletrônica de Varredura (MEV) e citotoxicidade in vitro. Através da determinação do conteúdo de elastina, foi possível determinar a massa média de colágeno presente nas matrizes, a qual foi de 95,2?0,2% (m/m), e a massa média de elastina, que foi de 4,8?0,2% (m/m), e também verificar que independente do tratamento, a elastina estava presente nas matrizes. O ensaio de estabilidade biológica mostrou que o tratamento com GA diminui a biodegradação do material; sendo obtidos porcentagens de degradação que variaram de 83,6%?1,1 (0% GA) a 46,1%?0,7 (0,085%-45min), indicando, assim, que com o aumento da concentração de GA e do tempo de reação, há uma diminuição da porcentagem de degradação. Pela análise termogravimétrica, foi observado que o colágeno presente nas matrizes tornou-se mais estável termicamente em conseqüência do aumento do grau de reticulação e, portanto, mais resistentes à degradação térmica. Os resultados de DSC confirmam os de termogravimetria devido ao aumento nos valores das temperaturas de desnaturação das matrizes em função do aumento do tempo de reação e da concentração de GA. Pela análise das fotomicrografias, pôde ser observado que após a reticulação com GA, as fibras de colágeno tornam-se mais organizadas e definidas; e essa definição torna-se maior com o aumento da concentração de GA. Os resultados de citotoxicidade in vitro mostraram que as matrizes analisadas são citotóxicas possivelmente devido a gordura remanescente, sendo necessário a realização de um pré-tratamento. Assim, a preparação de matrizes derivadas de pele porcina com diferentes tempos de degradação, as quais podem ser utilizadas na reconstrução de tecidos moles, é viável.
Cutaneous lesions and burns are considered the main causes of damage of soft tissues. In severe cases of trauma, the natural processes of regeneration are insufficient in the repair of the damage, resulting in chronic cutaneous lesions. Desvitalization of homologous or heterologous matrices is an alternative for the production of dermal matrices. The porcine skin is quite similar to the human skin and can be used as collagen matrix in soft tissue regeneration. Besides, it contains type I collagen as the main constituent and thus, it can be used in second degree burns. The objective of this work was the preparation and characterization of type I collagen extracellular matrices with alkaline hydrolysis and glutaraldehyde (GA) crosslinking. The collagen matrices were obtained from the alkaline hydrolysis of porcine skin, with subsequent GA crosslinking, in different concentrations (0 - 0,1%) and reaction time (15 and 45 min). Matrices were characterized by determination of the elastin content, biological stability (trypsin), differential scanning calorimetry (DSC), termogravimetry (TG/DTG), scanning electron microscopy (SEM) and a preliminar assay of in vitro cytotoxicity. Elastin and collagen content were 4,8±0,2% (m/m) and 95,2±0,2% (m/m), respectively. Biological stability results showed that GA crosslinking reduces matrix biodegradation; as degradation varied from 83,6%±1,1 (0% GA) to 46,1%±0,7 (0,085% - 45min), demonstrating, thus, that with the increase of GA concentration and reaction time, there was a decrease of degradation. For termogravimetric analysis it was observed that the collagen present in the matrices become termically more resistant as a consequence of the increasing crosslink degree and, therefore, more resistant to thermal degradation. DSC results, similar to termogravimetric ones, showed an increase in denaturation temperatures as a function of increasing reaction time and GA concentration. SEM analysis showed that after the GA crosslinking, collagen fibers become more organized and defined; and that definition improved with increasing GA concentration. Preliminar assay of in vitro cytotoxicity showed that treated matrices are cytotoxic possibly due to remaining fat, being necessary the accomplishment of a pre-treatment. Therefore, porcine skin matrices preparation with different degradation times, which can be used in the soft tissue reconstruction, are viable.

Currently, the in vitro production (IVP) of porcine embryos suffers from low efficiency and reduced blastocyst quality. Poor outcomes from in vitro matured oocytes and in vitro fertilised embryos have limited the use of assisted reproductive technologies (ARTs) within commercial porcine herds, reducing the potential for global genetic improvement programs. It is believed that this reduced developmental competency compared to in vivo embryos is attributable to altered metabolism resulting from in vitro culture. Improper or incomplete metabolic support from the culture media leads to production of inferior embryos. Much of the prior research centres on metabolism of carbohydrates by oocytes and embryos, with the formulation of media based on this knowledge. However, oocytes and embryos also contain endogenous lipid substrates, and there is a lack of understanding as to how and when these stores are utilised. Lipids are a dense form of energy storage, and there is evidence of their metabolism by oocytes and embryos for energy generation. Porcine oocytes and embryos have higher intracellular lipid content than other domestic livestock species, and this makes them an excellent model for studying aspects of lipid metabolism in vitro. The aim of this study was to examine the impact of lipid metabolism on the acquisition of developmental competence during porcine IVP, and how this is affected by the presence or absence of exogenous carbohydrates. Stimulation or inhibition of the β-oxidation pathway was used to discern the importance of fatty acid oxidation to oocyte maturation and embryo development during in vitro maturation (IVM), in vitro fertilisation (IVF) and in vitro embryo culture (IVC). During IVM, it was identified that porcine oocytes are capable of using different substrates to compensate for deficiencies in others. While pyruvate and glucose are preferentially utilised to support maturation, upregulation of β-oxidation can compensate for a low glucose concentration and an absence of pyruvate to support nuclear maturation. Although there was no discernible decrease in lipid content associated with this, lipids provide such a dense energy reserve that any usage may have been beyond the limit of detection. Inhibition of β-oxidation in the absence of carbohydrates had a greater effect on nuclear maturation compared to inhibition in complete media. This indicates that lipid metabolism plays a minor role during oocyte maturation in the presence of carbohydrates and is likely to be more important when other substrates are deficient. Energy generation prior to fertilisation is an important factor in the developmental outcomes of subsequent embryos. Upregulation of β-oxidation for the duration of IVF increased cleavage rates, but doses above 6mM L-carnitine led to decreased blastocyst development. This effect may be attributable to the antioxidant activity of L-carnitine, with low levels of reactive oxygen species (ROS) being required at fertilisation for normal sperm function and sperm-oocyte interactions. Oocyte incubation in media supplemented with 3mM L-carnitine for an hour prior to insemination increased cleavage and improved cryosurvival of Day 7 embryos after vitrification. While ATP content of oocytes did not increase over this period, it is unclear if lipid content was reduced. Previous studies have shown that L-carnitine treatment of oocytes and embryos decreased lipid content, thereby increasing cryotolerance. It would therefore appear that there is a limited role for β-oxidation during the IVF period itself, although upregulation immediately prior to fertilisation may have beneficial effects on metabolic processes and may provide antioxidant protection leading to improved development in early cleavage stage embryos. During embryo culture, there was a greater effect of upregulating lipid metabolism seen in the absence of carbohydrate substrates than in complete media. However, this could not support embryo development to the same extent as carbohydrate substrates. Changing nutrient requirements of embryos has led to the development of sequential media, leading to the production of better quality IVP embryos. Upregulation of β-oxidation for the first three days of culture in a single media system increased embryo quality to the same extent as a sequential carbohydrate media system, implying there is some level of plasticity to embryo metabolism allowing for adaptability to different substrates. Inclusion of L-carnitine for either a three day period or the duration of culture increased cryosurvival, suggesting decreased lipid content due to increased β-oxidation activity. Similarly for oocyte maturation, β-oxidation appears to be able to compensate for carbohydrate deficiencies during embryo culture to some extent, and oxidation of lipids has a greater role in promoting embryo quality over increasing production rates. The findings reported in this thesis represent a contribution to the understanding of lipid metabolism during the in vitro production of porcine embryos. These results provide evidence to support a level of adaptability of porcine oocytes and embryos to different substrates available during maturation and culture. There is a preference shown for carbohydrates substrates, with the ability to utilise lipids to compensate for certain deficiencies. This would justify the inclusion of co-factors of lipid metabolism such as L-carnitine in culture media, to ensure that any deficiencies in other substrates might be corrected for and to promote higher embryo quality. Upregulation of β-oxidation also increased the cryosurvival of porcine embryos following vitrification, with this being a major development in the global movement of superior genetics for herd improvement programs. These findings will also have implications for improving in vitro culture of oocytes and embryos of other species, most notably advancements in human ARTs where research is predominantly limited to work in animal models. The understanding of how lipids are metabolised alongside exogenous carbohydrates will contribute to improving media formulations for better metabolic support in vitro, further to improving embryo production and quality.

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses.

The failure behavior of collagenous soft tissues is important for clinical problems of plaque rupture and trauma. Cyclic tests require high frequencies that may affect the strength properties of the soft tissues. Experimental results of mechanical response of blood vessels to physiologic loads can be used to model and predict plaque rupture and direct medical therapy or surgical intervention. The goal of the study is to measure the mechanical failure properties of arteries to determine if they are strain rate and cycle dependant and to measure the progressive damage of arteries with time dependent loading. Ring specimens of porcine carotid arteries were preconditioned and then pulled to failure. In all cases, the intima broke first. Ultimate stress increased as a weak function of increasing strain rates. The ultimate stress at 100 mm/s was 4.54 MPa, greater than the 3.26 MPa at 0.1 mm/s. Strain rates between 1 and 100 mm/s correspond to a cyclic frequency of 0.5 Hz to 5 Hz for fatigue testing. In contrast, ultimate strain in arteries was independent of strain rate over the range tested. The creep tests showed a logarithmic relationship between stress magnitude and stress duration for this soft tissue. The creep testing indicates that damage is accumulating above certain threshold stress levels. The values of ultimate strength showed a 35% increase after 10,000 cycling loading. In contrast, the ultimate strain had a 13% decrease after cycling and the difference was statistically significant with p=0.018. The testing results showed that there were no significant differences on strength among fresh arteries and arteries stored at 5¡ã C for up to two weeks. The test results may be useful for developing a mathematical model to predict the behavior of arterial soft tissues and may be extended to estimate fracture and fatigue in the atherosclerotic plaque cap.

In this thesis we try to model the spatial pattern formed by slow oxidative (SO) fibres in pig muscle. Unlike the other mammals, the muscle fibre pattern in porcine muscle is characterised by cluster of SO fibres dispersed throughout the tissue. The biological interests of modelling such a configuration are, on one hand, its peculiarity, and, on the other hand, the effective contribution of such fibre patterns to the quality of pork meat. Transverse sections of muscle samples are stained to enable discrimination of the fibres of interest. The distribution of the SO clusters is investigated in low magnification cross-sectional images (x5). Each image is regarded as a spatial point process where points are the centroid of the corresponding clusters. Edge effects are minimised by discarding from the analysis those clusters that are touching the edges of the image. The minimum distance found between cluster centres suggests that clusters are distributed throughout the tissue according to some repulsive process. Higher magnification images (x10) are used to study SO fibre clusters in terms of their characteristics, namely the number of constituent fibres,  total area and shape. One arrangement of particular interest is that, given the number of constituent fibres, cluster cells are arranged in a concentric configuration. It is believed that the most centrally located fibre in a cluster corresponds to its respective primary fibre, the first constituent cell in any cluster. In this thesis, we suggest models to fit the porcine pattern of fibres based on the minimum inter-cluster distance.  All these models are based on using the minimum inter-primary distance established in the very beginning of the cluster process. These models vary essentially in the way in which cells are added to the primary fibre.  Basically, cluster cells are either (1) members of the primary’s neighbourhood or are (2) neighbour cells of any of the fibres in the cluster. For each of the methods described above we consider a nearest neighbour (NN) version, i.e., (1) from the primary’s neighbourhood cells are added in increasing order and (2) each new cell added to the cluster is the NN of a fibre of the cluster.

Animals are adapted to live in fluctuating environments. Some stimuli to which they are exposed will be ignored, some will be avoided and others will be approached. Stimuli perceived as a threat or associated with a painful stimulation will tend to be avoided. Therefore to understand more fully how an animal copes with a particular situation, e.g. transportation, its perception of all stimuli needs to be determined. The aim of the study reported in this thesis was to determine how auditory stimuli, to which pigs are exposed during production, are perceived by individual pigs. A field study was carried out to characterise the sounds to which pigs are exposed during production and studies were made of pig responses to sound under experimental conditions. The sound pressure level in artificially ventilated fattening units was quite loud (70 to 80 dB(Lin)), but relatively constant. In contrast, naturally ventilated units were quieter (60 to 70 dB(Lin)), but more variable. Sound pressure levels during transport were more than 88 dB(Lin) and highly variable. Similar levels were measured in articulated transporters and small livestock trailers. Sound pressure levels measured in abattoir lairages varied from 77 dB(Lin) to 89 dB(Lin). Equivalent sound pressure levels (Leq 20 min) of 97 dB(Lin) were measured in the stun pen of one abattoir that used electric stunning. Pigs' perception of mechanical sounds between 85 and 100 dB(Lin) was assessed. The onset of sound activity and visual searching. Stronger responses were measured for louder sounds. Over a constant exposure period of 15 to 20 minutes the responses observed decreased towards basal levels.

A number of different cell cultures were examined for their susceptibility to the influenza virus A/swine/Weybridge/86(H1N1) and A/swine/Weybridge/87(H3N2). PK1 (porcine kidney) was found to be the most susceptible to the viruses, and MDCK (canine kidney), the best cell line for primary isolation. A method of infectivity assay by immunoperoxidase in microplate cultures of MDCK cells was developed which was simple enough for routine use and practically as sensitive as the egg infectivity test. The potential risks of accidental importation of influenza infection in pig was assessed by determining the survival time of the porcine influenza virus H1N1 in pig tissues. It was found that the virus may keep its infectivity in frozen (-20°C) pig tissues for up to 15 days. The interspecies transmission of porcine influenza viruses was studied using turkeys infected with porcine influenza isolates. Although both A/swine/Weybridge/86 and A/swine/Weybridge/87 were transmitted from infected turkeys to pigs, only A/swine/Weybridge/86(H1N1) infected turkeys presented clinical signs of disease. More than 50% of the pigs presented the virus in the nostrils and/or faeces, at some time during the experiment, and all seroconverted. Transmission from these pigs to newly introduced turkeys was not observed, nor was seroconversion detected. Influenza epidemiology in Brazil was investigated by serological studies using pig sera collected in different areas of that country, using human, porcine and avian isolates of influenza viruses. Highest antibody titres were found against A/Leningrad/86(H3N2) (19%) and A/Port Chalmers/73(H3N2) (17%), but not against specific porcine isolates. Only serological evidence was found to suggest that reassortant influenza viruses occur in English pig herds. However, interspecies transmission of influenza viruses between man and pigs, and the maintenance of human strains in English pig herds was demonstrated by the isolation of two H3N2 influenza viruses very similar to A/Port Chalmers/73, present in the human population in the 1970s.

La present tesi doctoral tenia com a objectiu complementar el coneixement actual sobre l’eficàcia de la vacuna enfront a PCV2 en condicions d’infecció subclínica i explorar dos conceptes nous (en quan a estratègies de vacunació enfront a aquest patogen) que poguessin augmentar l’eficàcia d’aquest tractament. El primer estudi pretenia avaluar la possible interferència de la presència de diferents nivells d’anticossos d’origen maten (AOM) en el moment de la vacunació, en l’evolució del guany mig diari de pes (GMDP). En aquest estudi, només es va detectar una possible interferència en l’eficàcia de la vacuna sobre el GMDP, quan es va considerar la subpoblació d’animals amb els valors S/P més alts. Per tant, l’impacte d’aquesta possible interferència en condicions de camp es probablement negligible en la majoria d’animals i de les granges. En el segon estudi, es va avaluar la viabilitat d’eradicar la infecció de PCV2 en una granja convencional infectada subclínicament amb PCV2 mitjançant una estratègia de vacunació en massa. L’aplicació durant una any de la vacunació en massa enfront a PCV2 (sense implementar mesures específiques de maneig o de bioseguretat) no va ser capaç d’eliminar l’infecció per PCV2. De fet, un cop la vacunació es va aturar, el virus es va detectar de nou. De totes maneres, la disminució dels nivells d’anticossos i la no detecció del virus durant la segona meitat del període de vacunació en massa deixa entreveure que la eradicació de la infecció de PCV2 mitjançant un programa de vacunació més llarg i més extensiu podria ser possible.
This thesis aimed to complement the current knowledge on PCV2 vaccination efficacy under subclinical infection conditions and give new creative concepts (“thinking out of the box”) for future related studies. The first study had the objective to assess the putative interference of different maternally derived antibody (MDA) levels at the time of vaccination on the average daily weight gain (ADWG) evolution. In this study, an apparent interference of vaccine efficacy on ADWG was noticed only when a small subpopulation of pigs with the highest ELISA S/P ratios was considered, Therefore, the impact of this possible interference under field conditions is probably negligible for most of the animals and farms. In the second study, the feasibility to eradicate PCV2 in a conventional PCV2 infected farm by using a mass vaccination strategy was assessed.. One year period of mass PCV2 vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out PCV2 infection. Indeed the virus became detectable again when vaccination was stopped. However, the decreasing antibody levels and the lack of viral detection during the second half of the vaccination period shed a light on eradicating this virus by applying a longer term vaccination in a wider area would be feasible.

Master of Science
Department of Animal Sciences and Industry
Duane L. Davis
Culturing stem cells is usually done on tissue-culture treated plastic. Over time the cells change their gene expression and start to differentiate. Porcine umbilical cord (PUC) stem cells express the embryonic transcription factors Oct4, Nanog and Sox2 and changes in their expression may be useful for to evaluating culture-induced changes in the cells. We developed an extract of porcine Wharton’s jelly matrix (JMX) that may provide some characteristics of the stem cell niche located in the umbilical cord. Our extract used whole cords and enzyme digestion to simplify preparation of the product. We compare cells cultured on plastic to those grown on thin and thick gels of JMX in four experiments. In Exp 1a and b, growing PUCs on a thick JMX coating for 3(1a) or 4(1b) d increased the number of cells at the end of culture (P < 0.05) with minimal effects on gene expression. In Exp 2 we compared PUCs grown on thin and thick layered JMX with added collagen (+C) and to control cells. The JMX layers caused the cells to adopt a small, round shape and to form clumps or colonies during culture. No differences (P > 0.10) were seen between thin10 +C and control wells for viable and total cell counts but thick layered +C resulted in decreased numbers of viable cells compared to thin + C (P < 0.10) and control wells (P < 0.05). In a follow up experiment (Exp. 3) growing the PUCs mixed within, rather than plating on top of, a thick layer of JMX + C caused marked morphological changes with dense 3-dimensional structures formed. Exp 4 compared JMX allowed to gel for 10 (Thin10 +C) or 60 (Thin60 +C) min before the non-gelled fraction was removed. There were no effects on cell numbers at the end of culture (P > 0.10) but Sox2 expression was increased in Thin60 +C compared to controls on plastic (P < 0.05) and Thin10 +C (P < 0.10). In summary, JMX extracts change cell morphology and in some formats increased cell proliferation and may increase Sox2 expression. Further investigation is needed to fully understand the effects of JMX on PUCs.

A potencialidade do uso de uma matriz de colágeno e de um creme preparado pela mistura de um creme hidratante comercial com um gel de colágeno (90:10 m/m) foi avaliada no processo de cicatrização de feridas planas produzidas em pele de ratos. A matriz acelular de colágeno e o gel de colágeno foram obtidos a partir de serosa porcina por meio de um tratamento alcalino que não altera a estrutura do colágeno nativo e remove componentes celulares. Este estudo fez um comparativo macroscópico e histológico do processo cicatricial das feridas planas tratadas com soro fisiológico ou creme comercial (controles) e as tratadas com a sutura de uma membrana de colágeno (matriz) ou creme com colágeno. As feridas foram produzidas pela retirada de um flap de pele de 20 'MM POT.2' e receberam curativos diários. O material para histologia foi coletado nos 3º, 5º, 7º e 9º dias pós-cirurgia. Apesar de não ter havido uma acentuada diferença na cicatrização das feridas planas dos dois grupos de controle e no grupo que recebeu tratamento do creme contendo colágeno, a presença deste no creme indicou uma pequena diferença no grau de colageinização, o que demonstra serem válidas mais investigações nesta direção, buscando uma melhor proporção creme:gel e/ou diferentes concentrações para o gel de colágeno. A membrana demonstrou ser uma ótima opção para o reparo de lesões por ser de fácil obtenção e armazenamento, ter baixo custo e ser excelente para manuseio (maleável e resistente), além de atender às principais exigências mencionadas na literatura para qualquer curativo biológico oclusivo
The potentiality of the use of a collagen based matrix and of a cream prepared by the mixture of a commercial cream plus collagen (90:10 w/w) were evaluated in the healing process of rats’ skin. The acellular collagen matrix and the collagen gel were obtained by an alkaline treatment of porcine serosa which does not damage the native collagen structure and removes cellular components. This study compared by macroscopy analysis and histology the skin healing repair of wounds treated with physiological solution or commercial cream (control groups) and those treated with collagen based matrix suture or commercial cream plus collagen mix. The wounds were made by removing a skin flap with 20 'MM POT.2' and have received treatment every day. The material for histology was retired on the 3rd, 5th, 7th and 9th days after surgery. Even without an accentuated difference in the healing process of both control groups and the wounds treated with the commercial cream plus collagen, its presence in the cream showed a small difference of the collagen level in the new skin what validate more investigations on this way, searching better cream:gel proportion and/or different gel concentration. The matrix demonstrated to be a very good option to help wound healing because it is easily shelf able and obtainable, it has cheap cost and it is extremely nice to handle (resistant and manipulation able), besides to follow the main requirements present in the literature citation for any biologic occlusive dressing

A utilização e o desenvolvimento de biomateriais para a regeneração tecidual são de grande importância, principalmente para a área médica e odontológica. Matrizes de colágeno derivadas de tecidos de origem animal são utilizadas devido o colágeno apresentar características como biodegradabilidade e biocompatibilidade. Essas matrizes podem ser obtidas a partir de várias fontes, sendo uma delas o pericárdio porcino, que apresenta vantagens como grande disponibilidade, baixo custo, fácil obtenção e possibilidade de sofrer modificações químicas. Além disso, tecidos de origem suína são muito similares aos tecidos humanos, podendo ser utilizados para a produção de biomateriais para a regeneração de tecido mole. Este trabalho teve como objetivo a preparação de membranas acelulares derivadas de pericárdio porcino por hidrólise alcalina em diferentes tempos, para posterior utilização em engenharia de tecido. As membranas de colágeno foram obtidas por hidrólise alcalina de pericárdio porcino durante 4, 8, 12 e 24 h e caracterizadas por análise histológica, microscopia eletrônica de varredura (MEV), avaliação da citoxicidade in vitro, estabilidade biológica in vitro (colagenase), titulação potenciométrica, porcentagem de absorção de água, calorimetria exploratória diferencial (DSC), termogravimetria (TG), análise térmica dinâmico-mecânica (DMTA) e ensaios de tração. A análise histológica mostrou que após 4h de hidrólise as células foram removidas das membranas. A avaliação da citotoxicidade in vitro mostrou que as membranas preparadas neste trabalho não são citotóxicas. Os ensaios de estabilidade biológica in vitro por colagenase mostraram que as membranas hidrolisadas degradaram mais rapidamente que a não hidrolisada e, quando comparadas com matrizes derivadas de pericárdio bovino, as derivadas de pericárdio porcino foram mais resistentes à degradação por colagenase. A titulação potenciométrica possibilitou determinar o número de grupos carboxílicos das membranas e o incremento desses grupos por molécula de colágeno. Os resultados mostraram que houve um aumento no número de grupos carboxílicos tituláveis nas membranas hidrolisadas e, consequentemente, houve um aumento do número de cargas negativas incorporadas na molécula de colágeno. As membranas hidrolisadas apresentaram uma maior absorção de água, uma diminuição das temperaturas de desnaturaçãoe e menor estabilidade térmica em função do aumento do tempo de hidrólise. Os ensaios de tração mostraram que após a hidrólise alcalina as membranas apresentaram maiores valores de resistência à tração e que a deformação é dependente do tempo de hidrólise alcalina. Esses resultados mostraram que a preparação de membranas de colágeno derivadas de pericárdio porcino com diferentes tempos de hidrólise alcalina é um procedimento viável para ser utilizado na produção de biomateriais para engenharia de tecido.
The use and development of biomaterials for tissue regeneration are of great importance, especially for medical and dental care. Collagen matrices derived from animal tissues are widely used because collagen has characteristics such as biodegradability and biocompatibility. These matrices can be obtained from various sources, such as porcine pericardium, which is a tissue that can be used due its low cost, wide availability and because it can be chemically modified. Besides, porcine tissues are very similar to human tissue and can be used to produce biomaterials for soft tissue regeneration. The aim of this study was the preparation and characterization acellular membranes by alkaline hydrolysis of porcine pericardium. Membranes were characterized by histological analysis, scanning electron microscopy (SEM), in vitro cytotoxicity evaluation, in vitro biological stability (collagenase), potentiometric titration, water absorption percentage, differential scanning calorimetry (DSC), thermogravimetry (TG), dynamic mechanical thermal analysis (DMTA) and tensile tests. Histological analysis showed that after 4h of hydrolysis, cells were totally removed from matrices. In vitro cytotoxicity showed that all matrices prepared in this work are not cytotoxic. In vitro biological stability tests (collagenase) showed that the hydrolyzed membranes degraded more quickly than the non hydrolized matrix and more resistant to collagenase degradation when compared to matrices derived from bovine pericardium. The potentiometric titration allowed the determination carboxylic groups and the increase of these groups per collagen molecule. Hydrolyzed matrices had an increase in water absorption, a decrease in denaturation temperature and a small decrease in thermal stability with the increase of hydrolysis time. Tensile tests showed that after alkaline hydrolysis matrices showed higher tensile strength and the deformation was independent of the time of alkaline hydrolysis. These results showed that the preparation of collagen biological matrices derived from porcine pericardium with different times of alkaline hydrolysis is a viable procedure to be subsequently used in the production of biomaterials for tissue engineering.

Thesis (M.S.) -- University of Maryland, College Park, 2009.
Thesis research directed by: Dept. of Fire Protection Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Atherosclerotic plaque caps are composed of a composite soft tissue material that becomes subjected to cyclic loading under stenotic flow conditions. The cyclic loading causes the plaque cap to fatigue and eventually fail. The hypothesis of this work is that arteries and plaque caps may fatigue which may be predicted by a stress vs. number of cycles (S-N) curve. The S-N curve has not been determined for almost any biological soft tissue. The Specific Aim of the thesis is to quantify an S-N curve for normal arterial soft tissue collected from cyclic tension testing. Coronary arteries from porcine hearts will be tested as a material that closely models the plaque cap in non-linear elastic behavior. The S-N curve will be developed through failure testing with multiple cycles at stresses between 0.5 and 5 MPa.

Currently more than 300 million adults are obese and 1 billion are overweight throughout the world. Obesity is frequently accompanied by an array of health conditions such as hyperglycemia, hyperlipidemia, hypertension, cardiovascular diseases, and diabetes which are all considered to be part of what is now known as the metabolic syndrome. The role of adipose tissue as an endocrine organ has been emphasized by the characterization of its hormones: leptin, adiponectin and resistin. All three proteins regulate energy utilization. Over the past decade, leptin and resistin have also been shown to affect the reproductive system. This suggests that other adipocytokines, such as adiponectin, may also affect reproduction. This relationship was investigated using a porcine in vitro maturation system. When porcine cumulus oocyte complexes were matured in the presence of 30mug/mL of recombinant adiponectin an improvement in the meiotic maturation was observed. Moreover, maturation of denuded oocytes revealed that adiponectin acts through the cumulus cells to improve meiotic maturation of porcine oocytes. Finally, maturation of cumulus-oocyte complexes in the presence of MAPK pathway inhibitors suggested that adiponectin acts at or downstream of MEK1/2 and 38MAPK. This study shows, for the first time, an effect of adiponectin on porcine oogenesis. Further investigation will determine whether adiponectin also affects embryo development.

Peripheral nerve injuries can lead to a major loss of function and affect quality of life. Current autologous treatments and commercially available products such as nerve guide conduits all have limitations. The aim of this study was to develop biocompatible, non-immunogenic nerve grafts using low concentration SDS to decellularise porcine peripheral nerves. Initially the porcine nerve anatomy was defined. Acellular nerves were then used as the basis for an in vitro study of perfused flow within the tissue for the introduction of Schwann cells - as the delivery of these cells is reported to stimulate axon regeneration. Furthermore, an in vivo study using a rat sciatic nerve injury model was conducted to evaluate the regenerative capacity of acellular porcine grafts. Histology confirmed an absence of cells and retention of the native nerve histioarchitecture. DNA levels were reduced by >95 % throughout the decellularised tissue. Immunohistochemistry showed retention of important extracellular matrix proteins such as collagen, laminin and fibronectin. In vitro biocompatibility studies indicated the acellular nerves were not cytotoxic to human dermal fibroblasts and primary rat Schwann cells. Uniaxial tensile testing showed a significant increase in ultimate tensile strength (UTS) and strain between native and acellular nerve tissues. In addition, mechanical testing of the nerves revealed porcine peroneal nerves to have a higher UTS value in comparison to the porcine tibial nerves. Analysis of the nerves using transmission electron microscopy concluded that the mechanical properties of nerves could not be determined exclusively by their ultrastructure or collagen fibril diameter. Porcine acellular nerve grafts were used as an in vitro and in vivo model for the introduction of primary rat Schwann cells. The in vitro nerve model confirmed that reseeded Schwann cells under perfusion maintained their phenotype and had a lower rate of cell death when compared to static conditions. A rat sciatic nerve in vivo gap injury model demonstrated that porcine grafts were able to promote axonal regeneration; however, they were not as effective as the autologous graft. In summary, acellular peripheral nerve tissue was found to have excellent potential for development of a tissue engineered graft to aid peripheral nerve regeneration.

The polymerase chain reaction (PCR) assay was adapted and optimised for specific detection of Lawsonia intracellularis genomic DNA segment in swine faeces. Lawsonia intracellularis is the aetiological agent of porcine proliferative enteropathy (PPE) and the PCR represents the first diagnostic test suitable for ante-mortem use in affected swine. Various methods designed to extract bacterial DNA from faeces were evaluated to establish a convenient and optimum protocol. The PCR was utilised in pig challenge studies to investigate the excretion patterns of L. inracellularis in weaner pigs orally inoculated with pure cultures of L. intracellularis. This challenge work demonstrated that the PCR was a suitable tool for detection of infection, and indicated that individual animals could excrete L. intracellularis organisms for periods of up to ten weeks post-challenge. Such an excretion period has major implications for the transmission of organisms in the field. For example, if infected growers are still shedding L. intracellularis organisms upon entry to the breeding population, then this is a possible route for the transmission of disease to younger, susceptible pigs. A more extensive, two-part investigation of the epidemiological aspects of PPE in the field followed. The investigation comprised a farm sampling study and a questionnaire postal survey. In the farm sampling study, faeces samples were collected serially over a ten month period from breeding gilts and their litters. Samples were subjected to PCR for the detection of infection, allowing estimation of within-herd prevalence, as well as determination of possible transmission patterns. The assay successfully detected the presence of L. intracellularis in the weaners and/or growers of three of the five farms selected for this study. The within-herd prevalence for these age-groups ranged from 10 to 30%. The PCR also confirmed infection in several of the adult breeding boars and gilts.

For patients with end-stage bladder disease for which other treatment options have failed, the patient is treated surgically with either urinary diversion or bladder augmentation. Enterocystoplasty is the most common of these options, and it involves augmenting the bladder using a section of the patient's own intestine. However, there are several problems associated with the use of intestine for augmenting the bladder, and therefore an alternative augmentation material may be of benefit to patients. Numerous approaches have been used to develop tissue-engineered scaffolds capable of successfully augmenting bladders. Some of these approaches have involved the use of acellular tissue-derived materials, whereby the tissues are decellularised in order to remove immunogenic material and therefore prevent an immune reaction when transplanted allogeneically or xenogeneically. Decellularisation protocols typically involve a variety of chemical and physical processes which remove cells and other immunogenic material from the tissues. A protocol was previously developed to decellularise full-thickness porcine bladders. This material may have utility in bladder augmentation. The process involved distending the bladders with, and placing them in, a series of solutions. It was demonstrated that distending the whole organs was a necessary step in the decellularisation process. It was thought that this procedure applied biaxial strain to the wall of the tissue and reduced its thickness sufficiently for the solutions to penetrate the entire wall of the tissue. However, the method of distending whole bladders was not compatible with a scalable manufacturing process, and therefore the biomaterial was not able to be developed further. The overall aim of this project was to develop a novel method of manipulating bladder tissue which would enable bladder tissue to be decellularised in a way which would be compatible with a commercial manufacturing process. The original bladder decellularisation process used 500 ml of solutions to distend bladders. Preliminary experiments demonstrated that this volume was not always adequate to decellularise larger bladders. Filling experiments were performed to find relationships between bladder size and bladder capacity. A relationship between bladder capacity and bladder width*length was found to have a high correlation. Bladders were decellularised when filled to capacities calculated using the relationship. No signs of cellular material were observed in histological sections of these bladders, and DNA quantification indicated a removal of more than 99% of the DNA relative to native tissue. In order to determine the state of mechanical deformation of bladders during decellularisation, markers were placed on the surface of twelve bladders which were immersed in isotonic solution and slowly filled. Images taken of the bladders and markers during filling were used to calculate the strain of the tissues during the tests. The previously found relationships for bladder capacity were used to calculate the capacity of these bladders. The stress, strain and thickness of the bladders were calculated at the point the bladders were filled to their respective capacities. These strains were invariant with bladder capacity, and were equal to 2.0 and 1.4 in the circumferential and longitudinal directions respectively. Applying these strains to three bladders during decellularisation appeared to result in a complete removal of cellular material. It was thought that applying the required strains to bladder tissue deformed in a flat sheet configuration would be compatible with a manufacturing process. In order to apply biaxial strain to this highly compliant material, it was recognised that it would be appropriate to deform it using discrete points, placed along the edges of the tissue. The stretching of flat sheets of bladder using this method was modelled using finite element modelling to find an optimal stretching regime. The models demonstrated that deforming the tissue using five discrete points along each edge of the material would be adequate to ensure that the required strains would be applied to the tissue for decellularisation to occur. So that flat sheets of bladder could be decellularised, a piece of equipment was designed to hold pieces of bladder in the state of deformation which was previously modelled. The equipment took the form of a 3D-printed frame. A procedure was developed to stretch bladder tissue onto the frame. To test the hypothesis that bladder tissue could be decellularised in a flat sheet configuration, six bladders were stretched onto the frames and subject to the decellularisation process. Histological sections taken from decellularised bladder samples demonstrated a complete removal of cellular material, and a DNA extraction and quantification assay demonstrated that 99% of the DNA had been removed relative to the native controls. Bladders decellularised using the original process were transported in transport medium and processed within 4 h of bladder collection. A manufacturing process would require the tissue to be stored before processing. It was also recognised that it may not be necessary to transport bladders---which are destined for decellularisation---in transport medium, which was developed in order to maintain viable urothelial cells. To test the effects of freezing and transportation without transport medium, bladders were collected from the abattoir, transported without transport medium and subject to either one (six bladders) or two (six bladders) freeze-thaw cycles. Twelve fresh bladders were transported with transport medium. Bladders were immersed in solution and mechanically tested by distension, and their stress--strain curves calculated. There was no statistical difference between the toe region modulus and the transition stress of fresh, once-frozen and twice frozen bladders. There was a small but significant increase in the linear region modulus and transition stress of fresh bladders compared to the once- and twice-frozen bladders. No significant differences were found between once-frozen and twice-frozen bladders. To determine the effect of this revised transportation regime on bladder decellularisation, six bladders were transported without transport medium, subject to two freeze-thaw cycles and subject to the decellularisation process. Samples taken from these bladders for histological analysis and DNA quantification exhibited a complete removal of cellular material. In conclusion, this study demonstrated that applying suitable strains to flat sheets of bladder tissue was a viable method of deforming bladder tissue in order for it to be decellularised. Freezing the tissue up to two times before decellularisation resulted in some small but significant changes to the mechanical properties of the tissue, but did not affect the efficacy of the decellularisation process. It therefore may now be feasible to commercially produce decellularised full-thickness porcine bladder tissue.

Once an animal is harvested for meat, skeletal muscle attempts to maintain ATP at or near antemortem levels. To maintain ATP levels postmortem, stored glycogen is catabolized to produce ATP through glycolysis and possibly oxidative metabolism. Hydrolysis of the produced ATP acidifies muscle until an ultimate pH is reached. The ultimate pH of meat directly impacts the quality characteristics of color, texture, and water holding capacity. Therefore, our research intends to describe the contributions glycolysis and oxidative metabolism play in determining ultimate pH and fresh meat quality. Traditionally, glycogen content at death was thought to be responsible for dictating ultimate pH. This was especially true in oxidative muscle with limited glycogen stores. Yet, our research indicated that in the presence of excess glycogen, oxidative muscle maintains a high ultimate pH. Rather, pH inactivation of phosphofructokinase is responsible for terminating postmortem glycolysis and brackets ultimate pH between 5.9 – 5.5. Meat with a pH below this range is uncommon. However, AMPK γ3R200Q mutant pigs produce meat with an ultimate pH near 5.3. Due to lower AMP deaminase abundance in their muscle, AMP levels are elevated late postmortem. Because AMP is a potent activator of phosphofructokinase, the aberrant meat quality from AMPK γ3R200Q mutant pigs is caused by extended postmortem glycolysis. Combined, these data further our understanding of the factors that contribute to the formation of fresh meat quality. We also characterized AMPK γ3R200Q muscle by investigating antemortem skeletal muscle lactate transport. Lactate is transported in or out of tissues by proton-linked iii monocarboxylate transporters (MCTs). Previous reports indicated that acute activation of AMPK increased monocarboxylate transporter expression in skeletal muscle of other species. Yet, it was unknown the impact chronic activation of AMPK will have on MCT1, MCT2, and MCT4 expression in pigs. Compared to wild-type pigs, the longissimus lumborum of AMPK γ3R200Q pigs increased both MCT2 and MCT4 protein expression. Our data suggest glycolytic skeletal muscle from the AMPK γ3R200Q pigs has increased capacity for antemortem lactate export from muscle and possibly increased pyruvate transport into the mitochondria.
Ph. D.

Thesis (Ph. D.)--University of Sydney, 2000.
Includes tables. Title from title screen (viewed Apr. 22, 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Dept. of Animal Science, Faculty of Agriculture. Includes bibliography. Also available in print form.

Nipah virus (NiV) is a highly pathogenic and zoonotic paramyxovirus in the subfamily Paramyxovirinae, genus Henipavirus. The virus causes outbreaks of severe febrile encephalitis with a high mortality rate in humans, and of encephalitic and respiratory disease but with a low mortality rate in pigs. The innate immune response has a critical role in limiting viral infection by activating antiviral state and adaptive immune response. As pigs are able to overcome the infection with NiV, the working hypothesis was that IFN induced signaling pathways are not completely inhibited by NiV in infected porcine cells enabling an antiviral state to be established. Indeed, there was no block of eIF2α phosphorylation in porcine fibroblast (ST) and monocytic-like (IPAM) cells, and human fibroblast (MRC5) cells. To address the potential activation of an alternative IFN induced pathway, the MAPK signaling pathways were examined. The findings revealed that NiV infection triggers different kinetics of phosphorylation of ERK and p38 MAPK in the selected cell types. The data also indicates that p38 MAPK to be indispensable for NiV replication in vitro especially in immune cells. As the involvement of immune cells in viral spread and in immune modulation of porcine adaptive immune response were reported. The next hypothesis stated that NiV infects immune cells and affects the population frequencies of PBMC in pigs. In vitro, productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells, by recovery of infectious virus, anti-genomic RNA and detection of structural N and non-structural C proteins. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV. In NiV infected piglets, the expansion of the CD4+CD8- T cells early post infection was consistent with a functional humoral response. In contrast, significant drop in CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting vaccine studies that antibody development is a critical component of protective immune response. Thus, both aspects of innate and adaptive immune response are affected and contribute to NiV pathogenesis. These findings will help researchers to design and establish vaccination programs that would be more effective in pigs.

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease (PCVAD). Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV). Both PCV2 and PRRSV have caused devastating diseases in the swine industry worldwide, resulting in immense economic losses. One of the most common co-infections in the swine industry is PCV2 and PRRSV. The aim of this dissertation research is to explore different experimental approaches to develop novel vaccines against the two major pathogens affecting swine production and study the basic mechanisms that may be involved in viral pathogenesis. Two types of porcine circovirus (PCV), PCV1 and PCV2, have been identified thus far. PCV1, first identified as a contaminant of the PK-15 cell line, is non-pathogenic and has a low prevalence in swine herds. PCV2 is highly prevalent in most swine-producing countries and is associated with clinical PCVAD. The non-pathogenic PCV1 shares similar genomic organization with PCV2. Previously, it has been demonstrated that a genetically modified infectious chimeric PCV1-2a virus can tolerate up to a 27 aa insertion in the C-terminus of the ORF2 without affecting infectivity and produce a dual immune response against PCV2cap and the inserted epitope tag. Therefore, we evaluated the use of the non-pathogenic PCV1 wild-type (wt) virus and chimeric PCV1-2a vaccine virus (vs) to express four known B-cell epitopes of PRRSV. Peptide epitopes of PRRSV-VR2385, including GP2II (aa 40–51, ASPSHVGWWSFA), GP3I (aa 61–72, QAAAEAYEPGRS), GP5I (aa 35–46, SSSNLQLIYNLT), and GP5IV (aa 187–200, TPVTRVSAEQWGRP) were inserted in frame into the C-terminus of the ORF2 of PCV1wt as well as the PCV1-2avs. Four PCV1-PRRSVEPI chimeric viruses and four PCV1-2a-PRRSVEPI chimeric viruses were successfully rescued and shown to be infectious in vitro and co-expressed PCV1cap or PCV2cap with each specific PRRSV epitope. Two independent animal studies were conducted to evaluate whether the non-pathogenic PCV1 can serve as a vaccine delivery vector and whether the PCV1-2a vaccine virus can be used to develop a bivalent vaccine against both PCV2 and PRRSV. We demonstrated that three PCV1-PRRSVEPI chimeric viruses and two PCV1-2a-PRRSVEPI chimeric viruses were infectious in pigs. Importantly, we demonstrated that the PCV1-PRRSVEPI and PCV1-2a-PRRSVEPI chimeric viruses not only induced specific PCV1 or PCV2 IgG antibody but also specific anti-PRRSV epitope antibody responses as well. Regardless of the PCV backbone used, we showed that the PCV-PRRSV chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. These results provided a proof of concept for the potential use of the non-pathogenic PCV1 as a vaccine delivery system for PRRSV or other swine pathogens and the use of PCV1-2a vaccine virus to generate a bivalent vaccine against both PCV2 and PRRSV. PRRSV causes a persistent infection and immunosuppression. Immunomodulation of the host immune system is caused by modulation of numerous interleukins, such as type I interferons, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-12 (IL-12) in infected pigs. Antigen-presenting cells (APCs) are the first line of defense, and their infection plays an important role in innate-mediated immune regulation during early immune responses. Among the APCs, pulmonary alveolar macrophages (PAMs), pulmonary interstitial macrophages (PIMs), and dendritic cells (DCs) are the main targets for PRRSV replication. The role of PRRSV-DCs interaction is not fully understood, and current research focuses on the production and regulation of interferons through DC-SIGN receptors. In this study, we evaluated the immunomodulation of MoDCs by PRRSV through interactions with the pDC-SIGN receptor, by blocking pDC-SIGN with recombinant hICAM-3-Fc or anti-pDC-SIGN mAb. Our results indicate that recombinant hICAM-3-Fc enhances mRNA expression of proinflammatory cytokines and that anti-pDC-SIGN mAb inhibits mRNA expression of TNF-α and IL-1α and enhances the expression of IL-12 induced by PRRSV in MoDCs. The results will help understand the molecular mechanisms of PRRSV pathogenesis.
Ph. D.

La regulació de l’engreixament porcí i la qualitat de la carn són encara poc coneguts. Inicialment, vam investigar la variabilitat de gens candidats localitzats a regions QTL associades amb caràcters de qualitat de la carn i contingut i composició de greix intramuscular. Vam identificar polimorfismes en aquests gens candidats a partir de dades d’RNA-seq i seqüències de genoma complet (WGS) de cinc porcs Duroc. El genotip d’ATP1A2 va ser significativament associat a la conductivitat elèctrica (CE) del múscul longissimus dorsi (LD) a nivell cromosòmic. Els nostres resultats suggereixen que el gen ATP1A2 pot estar involucrat en la regulació de la CE en el múscul. D’altra banda, vam fer ús de les dades de WGS per localitzar mutacions stop gained segregant en una població Duroc (Lipgen). Van ser identificats set porcs homozigots per una mutació potencialment letal en el gen ASS1. Després de seqüenciar aquesta regió a nivell genòmic i transcriptòmic, es va revelar la presència d’una mutació immediatament abans, que eliminaria el codó d’aturada, generant un polimorfisme dinucleotídic que causa un canvi benigne d’aminoàcid en la seqüència d’ASS1. Seguidament, vam utilitzar dades prèvies d’expressió diferencial d’RNA-seq per investigar l’associació de gens candidats amb caràcters de qualitat de la carn. Dos polimorfismes localitzats en els gens CRY2 i MIGA2 van mostrar associacions significatives amb el contingut d’àcid esteàric en LD, i amb la concretació d’LDL en sèrum, respectivament. Aquests polimorfismes també van ser associats amb l’expressió dels seus gens respectius. Anàlisis a nivell cromosòmic van mostrar que aquests polimorfismes poden no ser els SNPs casuals. També vam analitzar els polimorfismes localitzats en gens microRNA a partir d’un total de 120 WGS de porcs domèstics i salvatges d’Àsia i Europa. La variabilitat de regions miRNA va ser molt reduïda en la seed comparat amb altres regions del miRNA i amb la resta del genoma. Quinze SNPs en gens miRNA van ser genotipats en la població Lipgen. Els resultats van revelar, entre d’altres, una variant localitzada en el bucle apical d’ssc-miR-326 significativament associada amb l’expressió d’alguns dels seus mRNAs diana. Aquest SNP pot contribuir a la reestructuració de l’aparellament de bases a la forquilla del miRNA, modificant l’eficiència de la maduració del propi miRNA. Altrament, ens vam proposar millorar l’encara limitada anotació del miRNAoma porcí mitjançant el desenvolupament d’un pipeline bioinformàtic per a la identificació i anotació de gens miRNA. La fracció d’RNA petit de 48 porques Duroc va ser seqüenciada per detectar miRNAs nous i ja coneguts. Els transcrits de dades d’small RNA-seq i miRNAs madurs anotats en humà van ser cartografiades en el genoma porcí. Es van reconstruir seqüències candidates mitjançant la cerca de motius nucleotídics. Es van obtenir un conjunt de paràmetres de seqüència i termodinàmics de cada seqüència i es va fer servir un algoritme de Machine Learning basat en grafs per predir miRNAs, tant nous com coneguts. Van ser descoberts un total de 47 miRNAs porcins putatius. L’expressió de tres d’ells va ser avaluada mitjançant tècniques d’RT-qPCR i confirmada en una població independent de porcs de raça Göttingen minipig. Finalment es van utilitzar les dades d’small RNA-seq per determinar miRNAs diferencialment expressats (DE) entre porques en dejú i després de rebre aliment. Les xarxes de regulació gènica d’interaccions miRNA-mRNA van revelar mòduls de coexpressió de gens relacionats amb el metabolisme de lípids. A més, es va evidenciar la potencial influència de miRNAs DE en regular l’expressió de mRNAs amb funcions en el metabolisme de la glucosa i l’homeòstasi energètica.
La regulación del engrasamiento porcino y la calidad de la carne son aún poco conocidos. Inicialmente, investigamos la variabilidad de genes candidatos localizados en regiones QTL asociadas con caracteres de calidad de la carne y contenido y composición de grasa intramuscular. Identificamos polimorfismos en dichos genes candidatos a partir de datos de RNA-seq y secuencias de genoma completo (WGS) de cinco cerdos Duroc. El genotipo de ATP1A2 fue significativamente asociado a la conductividad eléctrica (CE) del músculo longissimus dorsi (LD) a nivel cromosómico. Nuestros resultados sugieren que el gen ATP1A2 puede estar involucrado en la regulación de la CE en el músculo. Por otra parte, hicimos uso de los datos de WGS para identificar mutaciones stop gained segregando en una población Duroc (Lipgen). Siete cerdos homocigotos para una mutación potencialmente letal en el gen ASS1 fueron identificados. Tras secuenciar dicha región a nivel genómico y transcriptómico, se reveló la presencia de una mutación inmediatamente antes, que eliminaría el codón de parada, generando un polimorfismo dinucleotídico que causa un cambio benigno de aminoácido en la secuencia de ASS1. Seguidamente, utilizamos datos previos de expresión diferencial de RNA-seq para investigar la asociación de genes candidatos con caracteres de calidad de la carne. Dos polimorfismos localizados en los genes CRY2 y MIGA2 mostraron asociaciones significativas con el contenido de ácido esteárico en LD, y con la concentración de LDL en suero, respectivamente. Estos polimorfismos también fueron asociados con la expresión de sus respectivos genes. Análisis a nivel cromosómico mostraron que estos polimorfismos pueden no ser los SNPs causales. Además, analizamos los polimorfismos localizados en genes microRNA a partir de un total de 120 WGS de cerdos domésticos y salvajes de Asia y Europa. La variabilidad de regiones miRNA estuvo muy reducida en la seed comparado con otras regiones del miRNA y con el resto del genoma. Quince SNPs en genes miRNA fueron genotipados en la población Lipgen. Nuestros resultados revelaron, entre otros, una variante localizada en el bucle apical de ssc-miR-326 significativamente asociada con la expresión de algunos de sus mRNAs diana. Este SNP puede contribuir a la reestructuración del apareamiento de bases en la horquilla del miRNA, modificando la eficiencia de la maduración del propio miRNA. Además, nos propusimos mejorar la aún limitada anotación del miRNAoma porcino mediante el desarrollo de un pipeline bioinformático para la identificación y anotación de genes miRNA. La fracción de RNA pequeño de 48 cerdas Duroc fue secuenciada para detectar miRNAs nuevos y ya conocidos. Los transcritos de datos de small RNA-seq y miRNAs maduros anotados en humano fueron cartografiados en el genoma porcino. Se realizó la reconstrucción de secuencias candidatas mediante la búsqueda de motivos nucleotídicos. Se obtuvieron un conjunto de parámetros de secuencia y termodinámicos de cada secuencia y se empleó un algoritmo de Machine Learning basado en grafos para predecir miRNAs, tanto nuevos como conocidos. Un total de 47 miRNAs porcinos putativos fueron detectados. La expresión de tres de ellos fue evaluada mediante técnicas de RT-qPCR y confirmada en una población independiente de cerdos de raza Göttingen minipig. Finalmente se utilizaron los datos de small RNA-seq para determinar miRNAs diferencialmente expresados (DE) entre cerdas en ayunas y tras recibir alimento. Las redes de regulación génica de interacciones miRNA-mRNA relevaron módulos de co-expresión de genes relacionados con el metabolismo de lípidos. Además, se evidenció la potencial influencia de miRNAs DE en regular la expresión de mRNAs con funciones en el metabolismo de la glucosa y la homeostasis energética.
The genetic modulators of porcine fatness and meat quality traits, as well as their mechanisms of action, are still poorly understood. First, we investigated the variability of candidate genes located within QTL regions associated with meat quality traits and intramuscular fat content and composition. Polymorphic sites located at candidate genes were identified based on RNA-seq data and whole-genome sequencing of five Duroc boars. Significant association between ATP1A2 genotype and electric conductivity (CE) in the longissimus dorsi (LD) muscle, as well as in a chromosome-wide analysis, were revealed. Our results suggest that the ATP1A2 gene might be involved in the regulation of the CE of the skeletal muscle. Moreover, we employed whole-genome sequencing data from the five Duroc boars to identify putative stop gained mutations segregating in a Duroc population (Lipgen). Seven pigs homozygous for a potentially lethal nonsense recessive mutation in the ASS1 gene were detected. After sequencing such region at the genomic and transcriptomic levels, the presence of an additional polymorphism located immediately before the nonsense mutation that disrupts the stop codon was revealed, forming a dinucleotide polymorphism that causes a benign amino acid substitution in the ASS1 sequence. Furthermore, we used previous RNA-seq differential expression data to investigate the association of candidate genes with meat quality traits. Two polymorphisms located in the CRY2 and MIGA2 genes showed significant associations with stearic acid content in LD and with LDL serum concentration, respectively. These SNPs were also associated with the mRNA levels of the corresponding genes. Joint chromosome-wide association analyses showed that these polymorphisms are not the ones showing the most significant associations. We also studied polymorphisms residing in microRNA genes. A total of 120 whole-genome sequences from European and Asian wild boars and domestic pigs were used for variant calling analyses, and polymorphisms within miRNA loci were investigated. Variability within miRNA loci was strongly reduced in the seed region compared with the rest of the miRNA sequence and other regions in the genome. Fifteen SNPs mapping to miRNA genes were genotyped in the Lipgen population. Our results revealed, among others, one variant located in the apical loop of ssc-miR-326 as significantly associated with the expression of some of its targets. This SNP might contribute to a structural rearrangement of the miRNA hairpin pairing, thus modifying the efficiency of the miRNA maturation. Subsequently, we aimed to improve the yet poorly annotated porcine miRNAome by developing a bioinformatic pipeline for the discovery and annotation of miRNA genes. The small RNA fraction of 48 Duroc gilts was sequenced and used to detect novel and known expressed miRNAs. Small RNA-seq transcripts and annotated human mature miRNAs were mapped to the porcine genome. Reconstruction of candidate hairpin sequences was performed by applying a motif search correction approach. A series of sequence and thermodynamic features were obtained from each sequence and a Machine-Learning graph-based transductive algorithm was employed for predicting novel and annotated miRNA sequences. A total of 47 unreported putative porcine miRNAs were detected with this approach. The expression of three of the unreported miRNAs was assessed by using RT-qPCR analyses and their expression in an independent Göttingen minipig population was confirmed. Finally, we employed the muscle small RNA-seq data set to determine differentially expressed (DE) miRNAs between fasting and fed pigs. Gene regulatory networks for miRNA-mRNA interactions highlighted co-expression modules containing lipid-related genes. The potential influence of several DE miRNAs in regulating the expression of mRNA genes with key roles in glucose metabolism and energy homeostasis was evidenced.

The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.