The diagnosis of porcine circovirus-associated disease (PCVAD) in pigs requires the detection of characteristic clinical signs and pathological changes, and the detection of virus in tissues of affected pigs. To increase Australia’s capacity to independently diagnose PCVAD in Australia, techniques for the detection of Porcine circovirus type 2 (PCV2) infection in pigs were developed and are reported in this thesis. These techniques were applied to samples obtained from normal pigs and pigs with disease and confirmed the presence of PCV2 and PCVAD in the Australian pig herd. Viral DNA was detected in tissues of infected pigs by both standard PCR and real-time PCR techniques. The real-time PCR was more sensitive. While the conventional PCR was able to detect approximately 100 copies of the viral genome, the real-time PCR was able to detect 20 copies of the genome. An immunohistochemical (IHC) technique which was also developed enabled the visualisation of PCV2 antigen in fixed tissues of pigs with PCVAD. The techniques that were developed were applied to an examination of tissues from pigs affected by illthrift and increased weaner mortality in herds in South Australia, New South Wales and Western Australia. Lesions suggestive of the PCVAD postweaning multisystemic wasting syndrome (PMWS) were detected and virus antigen was detected in association with lesions. The nature of the clinical signs and histopathological lesions detected, coupled with the presence of PCV2 antigen, suggested that PCVAD was present in some Australian pig herds. Phylogenetic analysis of the strains of PCV2-associated with these disease outbreaks demonstrated they were of a type not previously detected in Australia and similar to strains associated with PMWS in North America. To further assist in investigation of PCV2 infections in the Australian pig herd, an enzyme-linked immunosorbent assay (ELISA) was developed that specifically detected antibody to PCV2 and not the related and non-pathogenic Porcine circovirus type 1. The development of this assay required the production of a virus capsid protein antigen using a prokaryotic protein production system. The ELISA was used to test serum samples form the Australian national pig serum bank. A high prevalence of PCV2 infection was detected in most pig herds examined in all Australian states. International trade in pig meat has resulted in many countries placing restrictions on the importation of pig meat, requiring imported pig meats to be cooked to destroy viral agents. This study investigated the in vitro resistance of an Australian strain of PCV2 to heat treatment at temperatures between 56°C and 85°C. The viability of the virus was determined by a combination of reverse transcriptase polymerase chain reaction (RT-PCR), and IHC to visualise viral capsid antigen within infected cells. This study indicated that PCV2 retained its infectivity following heating up to and including 75°C for 15 mins, but was inactivated following heating to 80°C and above. The investigations reported make a significant contribution to PCV2 research in Australia and ensure Australia’s capacity to independently investigate PCVAD in the Australian pig herd.

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded DNA virus that causes disease in pigs and is an economically important pathogen affecting pig populations worldwide. PCV2 contains two major open reading frames (ORF): ORF1 encodes two replicase proteins and ORF2 encodes the immunogenic capsid protein. There are three genotypes of PCV2 (PCV2a, PCV2b, and PCV2c), but vaccines available for PCV2 infection are only targeted against PCV2a. The objective of this thesis was to create viable chimeric PCV2 viruses with an ORF2 displaying genetic diversity from all PCV2 genotypes by synthetic DNA shuffling. Variation was identified at 55 amino acid positions in the ORF2 gene among 853 PCV2 capsid gene sequences available in the GenBank database. Degenerate oligonucleotide primers spanning ORF2 were synthesized to contain this naturally observed sequence diversity. Sets of overlapping oligonucleotide primers were fused together using overlap extension PCR to create full-length shuffled ORF2 sequences. The shuffled library of the ORF2 genes was subsequently cloned into the genomic backbone of a wildtype PCV2a infectious DNA clone and transfected into porcine kidney cells (PK-15). After transfection and infection of PK-15 cells, viability of chimeric viruses was screened by immunofluorescence assay (IFA) using anti-PCV2 Rep antibodies. PCR was used to amplify the genomes of viable shuffled viruses from infected cells. PCV2 viruses containing an ORF2 displaying genetic diversity from PCV2a, PCV2b, and PCV2c were isolated in vitro. These shuffled PCV2 viruses may be used as potential candidates for a broadly-protective PCV2 vaccine, although additional studies are warranted to determine in vivo infectivity and pathogenicity. / Master of Science

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated disease (PCVAD), an economically important swine disease that causes wasting in pigs 5-18 weeks of age. There exist two different types of porcine circoviruses: porcine circovirus type 1 (PCV1) was discovered as a contaminant of porcine kidney (PK-15) cells and was determined to be nonpathogenic in swine; whereas porcine circovirus type 2 (PCV2) is pathogenic. A recently released vaccine for PCVAD was generated by inserting the gene encoding the immunogenic capsid protein of PCV2 into the genetic backbone of the non-pathogenic PCV1. This chimeric PCV vaccine, called PCV1-2, was shown to induce protective immunity against PCV2 infection in pigs. The vaccine is currently on the market in a killed form. In order to develop a live version of the vaccine, the genetic stability of the chimeric PCV1-2 vaccine virus was investigated by in vitro and in vivo passaging of the vaccine virus. In vitro passaging of the PCV1-2 vaccine virus was done in a porcine kidney PK-15 cell line. Cells were infected with the PCV1-2 vaccine virus and then serially passaged 11 times. The passaged vaccine viruses recovered from passages 5 and 11 were sequenced, and the sequences were compared to that of the original PCV1-2 vaccine virus. The in vitro serial passage result showed that no mutation occurred during the 11 in vitro passages. The in vivo passaging was done using specific-pathogen-free (SPF) pigs. In in vivo "passage 1", nine piglets were divided into 3 groups of 3 each: group 1 each inoculated with 200ug of PCV1-2 plasmid, group 2 each with 1Ã 103 TCID50 live PCV1-2 vaccine virus, and group 3 each with 3ml phosphate buffered saline (PBS) buffer as a control. One pig from each group was necropsied at 14, 21, and 28 days post-inoculation (DPI), respectively. A panel of tissue samples including lymph nodes and thymus were collected from each pig. Tissue homogenates from DPI 28 that were positive by PCR for PCV1-2 DNA were used to inoculate new piglets in the in vivo passage 2 experiment. Viruses recovered from passage 2 pigs were subsequently used for inoculation in the in vivo passage 3 experiment. The PCV1-2 vaccine virus DNA from pigs in each passage was amplified and sequenced. The results of the in vivo serial passage experiment showed that, after 3 passages of the PCV1-2 vaccine virus in pigs, there were no new mutations in the viruses recovered from pigs. The PCV1-2 vaccine contained an introduced marker mutation at amino acid position number 79, which is in the capsid region. During the in vivo passaging of the vaccine virus in pigs, this marker mutation quickly reverted back to its original nucleotide. This marker back mutation occurred between DPI 21 and DPI 28 of passage 1 in the PCV1-2 live vaccine virus group, and between DPI 28 of passage 1 and DPI 14 of passage 2 in the PCV1-2 vaccine plasmid group, and remained stable throughout the reminder of the in vivo study. Based upon the results from this study, we conclude that the PCV1-2 chimeric vaccine virus is genetically stable in vitro and in pigs, and thus should serve as a good candidate for a live vaccine against PCV2. / Master of Science

Circoviren sind kleine, unbehüllte Viren mit einem einzelsträngigen zirkulären DNA Genom mit eine Größe von 1,7 bis 2,4 kb. Das Porcine Circovirus Typ 2 (PCV2), welches zum Genus Circovirus gehört, ist mit einer Anzahl von Krankheitsmanifestationen verbunden worden, die heute als Porcine Circovirus Assoziierte Krankheiten (PCVAD) zusammengefasst sind. Die PCV2-Infektion bei Rindern ist bis zum jetzigen Zeitpunkt marginal erforscht worden. Serologische Untersuchungen auf Circovirus spezifische Antikörperführten zu widersprüchlichen Ergebnissen. Im Jahr 2007 wurde von der Bovinen Neonatalen Panzytopenie (BNP) in Europa mit unklarer Genese berichtet. Das klinisch - pathologische Bild der Hämorrhagien ähnelte dem Krankheitsbild der Infektiösen Anämie, welche durch ein Circovirus bei Hühnern verursacht wird. Deshalb wurde in dieser Studie eine Breitspektrum PCR zum Nachweis von Cirocvirus-Genomen durchgeführt. In 5 von 25 BNP betroffenen Kälbern konnte circovirale DNA nachgewiesen werden. Das komplette Genom wurde nachfolgend amplifiziert, kloniert und sequenziert. Das nachgewiesene Genom (PCV2-Ha08) hat eine Länge von 1768 Nukleotiden und zeigte eine hohe Homologie (bis zu 99%) mit PCV2-Genotyp b (siehe Publikation 1). Als Ursache der BNP ist vor kurzen die Übertragung von Alloantikörpern über das Kolostrum beschrieben wurden, welche die Zerstörungen von Leukozyten und Thrombozyten sowie deren Vorläuferzellen bewirken. Ungeachtet dessen war es wichtig, die Empfänglichkeit und Immunantwort von Kälbern nach experimenteller Infektion mit PCV2 zu studieren. Für diesen Zweck wurden weitere 181 Proben von BNP-Kälbern aus Deutschland mit Hilfe einer Breitspektrum-PCR getestet. In zwei von 181 Proben wurde PCV2 DNA nachgewiesen. Die vollständigen Sequenzen konnten amplifiziert werden. Während das erste Genom aus einer Blutprobe eines Kalbs in Bayern stammte (PCV2-Ha09), stammte das zweite nachgewiesene Genom aus Lunge und Gehirn von einem Kalb in Sachsen (PCV2-Ha10). Das Genom (PCV2-Ha09) besteht aus 1768 nt, währenddessen das Genom (PCV2-Ha10) aus 1767 nt aufgebaut ist (siehe Publikation 2). Weiterhin wurden die PCV2 Empfänglichkeit und die Immunantwort von Kälbern durch experimentellen PCV2 Inokulation sowie die Möglichkeit, eine Serokonversion nach Impfung mit einer kommerziellen PCV2 Vakzin zu entwickeln, untersucht. PCV2-spezifische Antikörper wurden in den PCV2-infizierten Tieren und in den PCV2-immunisierten Tieren im Tag 11 und 7 nach Inokulation (p.i.) nachgewiesen. PCV2-Genome wurden durch quantitative Realtime-PCR zwischen Tag 4 und Tag 46 p.i. nur in den Blutproben sowie in verschiedenen Geweben (z.B. Milz, Lymphknoten, Thymus) der PCV2-infizierten Tiere nachgewiesen. Das Genom, welches von den Lymphknoten der PCV2-infizierten Kälber erneut isoliert wurde, zeigt eine Identität von 99,9% gegenüber dem Inokulum. Dies weist möglicherweise auf adaptierte Mutationen im PCV2 Genom hin. Die Mutationen C1708T und G365C sind während der Infektionen aufgetreten. Die Sequenzanalyse zeigt eine mögliche adaptierte Mutation an der Aminosäure Nr. 105 in Replikationsgen (Met zu Ile) (siehe Publikation 3). Zusammenfassend kann geschlussfolgert werden, dass der Nachweis der PCV2 Genomen und eine experimentell induzierte Serokonversion möglich war. Es konnte gezeigt werden, dass die Empfänglichkeit von PCV2 nicht allein auf Schweine begrenzt ist und eine Übertragung von PCV2 auf Rinder möglich ist.

Made available in DSpace on 2015-03-26T13:47:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 914989 bytes, checksum: 6324bfc7a5398520ac5042a3539b706e (MD5) Previous issue date: 2011-11-22 / Porcine circovirus-2 (PCV2) has been related as the causative agent of the Postweaning Multissystemic Wasting Syndrome (PMWS) and other diseases called porcine circovirus associated diseases (PCVAD). They are responsible for economic losses in pork production worldwide. There is only a few scientific studies describing the infection in other species but swine and their hole at the epidemiologic dynamics of the diseases related to the PCV2. The aim of this study is to investigate the occurrence of infection by the PCV2 in wild mice (Mus musculus and Rattus rattus) captured in hog farms. The capture of the 40 sorted mice was made at 5 pig wean tofinish farms in Minas Gerais, an important state of pork production in Brazil. Samples of tissues (lymph nodes, spleen, kidney, heart and lungs) and blood were collected from the mice. The tissue fragments collected were submitted to immunohistochemistry and Nested PCR. Additionally, samples from spleen and lungs were analyzed by histology assays. Presence of antibodies anti-PCV2 was tested by ELISA assays. Immunohistochemical analysis showed positive prints in 12 animals, mostly on spleen (sub scapular area), lungs (alveolar macrophages) and kidney (inside the tubules). The 12 serum analyzed by ELISA hasn t detected antibodies anti-PCV2. Histopathological analyses revealed in some samples, a multifocal and lympho-neutrophilic interstitial bronchopneumonia, with some node formations. Moreover, spleen samples showed a mild to moderate lymphocyte depletion related to the PCVAD. The Nested PCR assays showed the presence of viral DNA at different tissues from 6 tested rodents. Thus, the results found in this work, indicate that mice from the species Mus musculus and Rattus rattus can be naturally infected by the PCV2 and they would play a hole in the epidemiology of PCVAD. However, more studies are necessary to confirm the transmission of the PCV2 from wild rodents to pigs. / O porcine circovirus-2 (PCV2) é atribuído como um dos agentes relacionados a doenças associadas ao circovírus (PCVAD), ocasionando perdas econômicas significativas na produção mundial de suínos. Poucos trabalhos são realizados a respeito da infecção em outras espécies pelo PCV2 e sua participação na epidemiologia das doenças associadas ao vírus. O propósito desse estudo foi investigar a ocorrência de infecção em roedores peridomésticos das espécies Mus musculus e Rattus rattus pelo PCV2 em granjas comerciais de suínos. Animais dessas espécies foram capturados em importantes centros de produção no estado de Minas Gerais. Amostras de órgãos (linfonodos, baço, rins, fígado, pulmão) e sangue foram coletadas. Os fragmentos de tecidos coletados foram submetidos ao teste de imunohistoquímica e Nested PCR. Adicionalmente, foram realizadas avaliações histológicas em amostras de baço, rim e pulmão. Presença de anticorpos anti-PCV2 foram avaliados pela técnica de ELISA. O teste de imunohistoquímica demonstrou marcações encontradas em 12 animais, principalmente no baço (região subcapsular), no pulmão (macrófagos alveolares) e nos rins (interior dos túbulos). A análise do soro pela técnica de ELISA não detectou anticorpos contra o PCV-2 nas 12 amostras avaliadas.. A histopatologia demonstrou em algumas amostras, uma pneumonia bronco-intersticial neutrofílica e linfocítica, multifocal e moderada, com formação de nódulos linfóides associados a vasos e bronquíolos. No ensaio de nested-PCR foi detectado DNA viral em diferentes tecidos avaliados de seis animais. Os resultados citados demonstram que os roedores domésticos das espécies estudadas podem exercer importante papel na epidemiologia das doenças relacionadas ao PCV2. No entanto, mais estudos são necessários para comprovar a transmissão do PCV2 dos roedores para os suínos.

Made available in DSpace on 2015-03-26T13:47:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 926741 bytes, checksum: 18d302c22a4907755b01b95cb9c1ea36 (MD5) Previous issue date: 2013-06-04 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The diseases associated with PCV2 (PCVAD) require various factors to occur, however, the virus infection is critical to the development of any one of the syndromes. The present study aimed to determine differences in serologic and viremic profiles for PCV2 in the swine breed Piau and a commercial line (Landrace x Large White x Pietrain) at a subclinically infected farm by the virus studied. The experiment was conducted at the Genetic Improvement Pig Farm (GMS), Federal University of Viçosa (UFV), in which it isn´t carried out vaccination against PCV2. This study conducted a cross-sectional sample of sows (> 2 parity), pigs for 1-3 weeks, 3-8 weeks and 8-22 weeks of age. The serum samples were used to measure the level of total antibodies by ELISA and quantitation of viremia by real time PCR. The results showed that, at the age of 3-8 weeks, the Piau breed piglets seroconverted earlier than the commercial line piglets and the Piau breed sows showed lower levels of total antibodies in relation to the commercial line. There were no differences in viremia between the different stages of production within each genetic group or between groups. This work provides evidence that the breed Piau has a different humoral immune response than the commercial line studied when facing a natural PCV2 subclinical infection. The results of this study reinforce the importance of the conservation of native breeds that have not been used for development of high productivity commercial lines. / As doenças associadas ao PCV2 (PCVAD) necessitam de vários fatores para ocorrer, no entanto, a infecção pelo vírus é fundamental para o desenvolvimento de qualquer uma das síndromes. O presente estudo teve como objetivo verificar diferenças nos perfis sorológicos e virêmicos para o PCV2 entre suínos da raça Piau e de uma linhagem comercial (Landrace x Large White x Pietrain) em uma granja subclinicamente infectada pelo vírus estudado. O experimento foi realizado na Granja de Melhoramento Genético (GMS) da Universidade Federal de Viçosa (UFV), na qual não é realizada a vacinação contra o PCV2. O presente estudo realizou uma amostragem transversal em porcas (>2º parto), suínos de 1-3 semanas, 3-8 semanas e 8-22 semanas de idade. As amostras de soro obtidas foram utilizadas para mensuração dos níveis de anticorpos totais por ELISA indireto e quantificação da viremia por PCR em tempo real. Os resultados demonstraram que, na idade de 3-8 semanas, os leitões da raça Piau soroconverteram mais precocemente em relação aos leitões da linhagem comercial e as porcas da raça Piau apresentaram menores níveis de anticorpos totais em relação às da linhagem comercial. Não houve diferença na viremia entre as diferentes fases de produção dentro de cada grupo genético ou entre os grupos. Este trabalho fornece indícios de que a raça Piau apresenta uma resposta imune humoral diferente da desenvolvida pela linhagem comercial estudada diante de uma infecção subclínica natural pelo PCV2. Os resultados obtidos neste estudo reforçam a importância da conservação das raças nativas que não foram utilizadas para formação de linhagens de alta produtividade.