SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts

Abstract Background Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts. Methods To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays. Results Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells. Conclusions Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.

Avaliação da morfometria renal através do método ultrassonográfico de fêmeas caprinas (Capra hircus) normais da raça Saanen

A utilização de técnicas ultrassonográficas na área de Medicina Veterinária está cada vez mais presente e a capacitação nesta área tornou-se indispensável para o clínico, auxiliando-o na obtenção de informações rápidas e valiosas das afecções patológicas que podem acometer os animais. O exame ultrassonográfico renal revelou-se de grande importância neste âmbito, com o objetivo de avaliar e mensurar os parâmetros morfométricos renais normais de fêmeas caprinas (Capra hircus). Por meio de técnicas ultrassonográficas procedeu-se o estudo de 30 fêmeas da raça Saanen, divididas em três grupos: fêmeas com idade inferior a 6 meses (3,0±1,0 meses), de 6-18 meses (9,0±4,3 meses) e com idade superior a 18 meses (46,3±17,4 meses). Realizaram-se imagens dos rins, em secções longitudinais, medidas de comprimento e largura e, em secções transversais, medidas de altura (ou espessura). Com estes valores calcularam-se volumes renais, corticais e medulares, além da relação cortico-medular. Com relação ao comprimento renal os grupos com idade inferior a 6 meses, de 6-18 meses e com idade superior a 18 meses obtiveram média e desvio padrão de 4,20±0,36cm, 5,56±0,40cm e 6,77±0,64cm, respectivamente. Tratando-se do volume renal, estes grupos apresentaram média e desvio padrão de 17,02±3,99cm³, 19,99±5,86cm³; e, 41,23±13,05cm³. Comparou-se a equivalência métrica das médias entre os dois rins de forma que os parâmetros volumétricos e lineares renais com diferença entre si são comprimento renal, volume renal e volume cortical para o grupo de fêmeas com idade de 6-18 meses, e comprimento renal e comprimento medular para o grupo com idade superior a 18 meses. Entre diferentes grupos observou-se que somente o comprimento medular esquerdo apresentou média equivalente em todos os grupos, ou seja, entre o grupo com idade inferior a 6 meses e o grupo com idade de 6-18 meses e, entre este último e o grupo com idade superior a 18 meses. Os resultados mostraram correlações diretas e positivas entre peso corporal e idade com os parâmetros lineares e volumétricos, a relação cortico-medular esquerda foi a única que apresentou correlação significativa com o peso (r= -0,365; P = 0,047). Para aqueles parâmetros que apresentaram correlação significativa foi realizada análise de regressão, obtendo-se a linha de melhor ajuste das variáveis.

Prevalência de leptospirose em caprinos leiteiros do semiárido paraibano

Este trabalho teve como objetivos determinar a prevalência de propriedades de caprinos leiteiros positivas e de animais soropositivos para leptospirose no semiárido paraibano. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos para a infecção por Leptospira spp. No total, foram colhidas amostras de sangue de 975 animais procedentes de 110 propriedades leiteiras localizadas no Munícipio de Monteiro, microrregião do Cariri Ocidental, Estado da Paraíba. Para o diagnóstico da infecção por Leptospira spp., foi utilizado o teste de soroaglutinação microscópica (SAM), utilizando como antígenos 24 sorovares. Uma propriedade foi considerada foco quando apresentou pelo menos um animal soropositivo. As prevalências de propriedades positivas e de animais soropositivos foram de 43,6% (IC 95% = 34,2-53,4%) e de 8,7% (IC 95% = 5,7-12,9%), respectivamente. Nos animais, os sorovares mais frequentes foram Autumnalis (1,74%; IC 95% = 0,97-3,09%), Sentot (1,71%; IC 95% = 0,82-3,52%) e Whitcomb (1,39%; IC 95% = 0,65-2,93%), e nas propriedades, os sorovares Autumnalis (10,9%; IC 95% = 5,8-18,3%), Whitcomb (8,2%; IC 95% = 3,8-15,0%) e Sentot e Patoc (7,3%; IC 95% = 3,2-13,8%) foram os mais frequentes. Sugere-se que a infecção está distribuída em caprinos da região, e que há necessidade de implantação de medidas de controle e prevenção, com o intuito de reduzir a ocorrência da infecção e, consequentemente, diminuir perdas econômicas ocasionadas e bloquear a possível transmissão do agente aos seres humanos.

Fagocitose intensificada de Corynebacterium pseudotuberculosis por células da série monócito-macrófago de caprinos naturalmente infectados pelo vírus da artrite encefalite

A Artrite Encefalite Caprina (AEC) e a Linfadenite Caseosa (LC) possuem alta incidência e transmissibilidade em pequenos ruminantes. Como ambas possuem tropismo por monócitos-macrófagos e afetam mecanismos da resposta inata do hospedeiro, acredita-se que a AEC predispõe o animal a infecções por Corynebacteruim pseudotuberculosis, agente etiológico da LC. Para confirmar esta hipótese, avaliou-se a fagocitose de células da série monócito-macrófago de cabras naturalmente infectadas pelo vírus da AEC (VAEC). Para tanto, foram utilizadas 30 cabras da raça Saanen, alocadas em dois grupos distintos, com 15 animais cada, conforme a sororreatividade de anticorpos séricos antivírus da AEC. Células mononucleares de sangue periférico foram isoladas por gradiente de densidade e plaqueadas para isolamento de células da série monócito-macrófago. Posteriormente, o ensaio de fagocitose de C. pseudotuberculosis foi realizado, após incubação por duas horas a 37ºC a 5% de CO2, e a visualização da fagocitose foi identificada por microscopia óptica. O presente estudo não encontrou diferença na porcentagem de monócito-macrófagos que realizaram fagocitose entre os diferentes grupos (P = 0,41). Todavia, a análise quantitativa de bactérias fagocitadas, demonstrou maior capacidade fagocítica pelos macrófagos-monócitos do grupo sororreagente ao vírus da AEC. Correlação entre monócitos fagocitando e macrófagos que fagocitaram mais de 12 bactérias foi observado neste grupo (r = 0,488; P = 0,006), não sendo o mesmo encontrado no grupo de animais sorroreagentes negativos. Os dados demonstram aumento na intensidade da fagocitose de macrófagos de animais infectados com o vírus da AEC.

Contagem de células somáticas e produção leiteira em cabras Saanen estressadas via aplicação de ACTH

Avaliou-se neste estudo o efeito do estresse, via administração de ACTH (hormônio adrenocorticotrófico), sobre a quantidade e qualidade do leite produzido e a indicação de estresse pela alteração no nível de cortisol. Assim, 12 cabras lactantes foram distribuídas em dois grupos: seis animais receberam aplicação de 0,06 UI de ACTH/kg de PV (desafiadas) e seis receberam solução placebo (controle), todas via intravenosa. O desafio ocorreu ao longo da lactação, com aplicação do protocolo aos 30, 60, 120 e 180 dias do início da lactação, avaliando-se em cada data os níveis de cortisol 30 minutos antes, no ato da aplicação, 60, 120 e 300 minutos depois. A produção leiteira foi mensurada diariamente durante 270 dias e, a cada 20 dias, o leite foi colhido para contagem de células somáticas (CCS) pelo método direto. Os teores de cortisol mensurados em -30 e 0 minuto antes da aplicação de ACTH foram baixos e semelhantes entre os grupos. Entretanto, a partir de 60 minutos, constatou-se efeito da aplicação de ACTH, com teor de cortisol de 61,37±6,65 versus 5,47±1,21 ng/mL e de 51,17±10,21 versus 4,67±1,21 ng/mL aos 120 minutos. Aos 300 minutos, o cortisol retornou ao nível basal. Não houve diferença na produção de leite, no teor de proteína e de gordura do leite, cujos valores nos grupos ACTH e Placebo foram 1,37±0,59 e 1,38±0,63 kg de leite, respectivamente. A contagem de células somáticas não foi influenciada pelo estresse e apresentou valores situados na faixa indicada para animais sadios. Constatou-se estresse pontual em decorrência da aplicação de ACTH, porém sem alterações de natureza quantitativa e qualitativa do leite produzido, e isso indica que atividades de manejo que estressam os animais pontualmente não trazem prejuízos ao sistema produtivo.

Parâmetros genéticos para a produção de leite em caprinos das raças Saanen e Alpina

Objetivou-se com este trabalho estimar parâmetros genéticos para a produção de leite acumulada até os 305 dias (P305) de cabras das raças Saanen e Alpina. Foram utilizadas as duas primeiras parições de cabras pertencentes a rebanhos participantes do programa de controle produtivo e reprodutivo de caprinos (PROCAPRI) da UNESP-FCAV-Jaboticabal-SP. A P305 foi analisada por meio de modelos de repetibilidade e bicaracterísticas. Para verificar a influência dos efeitos fixos sobre a característica analisada foram realizadas análises preliminares, pelo método de quadrados mínimos. Os componentes de covariâncias foram estimados pelo método da máxima verossimilhança restrita (REML), utilizando o programa Wombat. A duração da lactação, a idade da cabra ao parto, o rebanho, o ano de parto e a estação de parto foram importantes fontes de variação para a P305. Não houve diferença significativa entre as raças estudadas. As estimativas de herdabilidade e repetibilidade para a P305, obtidas com o modelo de repetibilidade, foram de 0,29 e 0,36, respectivamente. As estimativas de herdabilidade, obtidas pelos modelos de repetibilidade e bicaracterísticas foram semelhantes. Sendo assim, um modelo de repetibilidade poderia ser indicado para avaliar a P305 pela sua simplicidade.

Morfologia do funículo espermático em caprinos da raça Bhuj Brasileira

Estudamos em 40 caprinos adultos da raça Bhuj Brasileira os aspectos histológicos do funículo espermático. Observamos que este se acha envolvido por uma cápsula de tecido conjuntivo fibroelástico denso, de espessura variável, pregueada em alguns pontos, e revestida por mesotélio que circunda todo o conjunto vásculo-nervoso, e projeta-se para formar o mesoducto deferente. Em posição subcapsular, verifica- se uma camada de tecido conjuntivo fibroelástico frouxo, de espessura variável, que circunda parcialmente o funículo espermático, isolando nas regiões deferencial e abdeferencial, conjuntos vásculo-nervosos, responsáveis pela nutrição do epidídimo. Na região do mesoducto deferente, o tecido subcapsular acompanhado de tecido adiposo constitui a camada interna deste meso, formando a sua adventícia e abrigando vasos e nervos deferenciais. Na região abdeferencial, pequenos acúmulos de tecido adiposo são vistos de permeio aos vasos e nervos desta região. Entre as artérias, veias e nervos testiculares, bem como entre os vasos das regiões deferencial e abdeferencial, observa-se o tecido conjuntivo denso, intervascular, rico em fibras elásticas, que constitui as adventícias contínuas destes vasos. O arranjo vascular mostra que o segmento da artéria testicular, contido no funículo espermático, apresenta trajeto sinuoso. Estando envolvido pelo plexo venoso pampiniforme, formado por veias testiculares desprovidas de válvulas de calibres variados, apresentando amplas comunicações entre si. As veias responsáveis pela drenagem do epidídimo e ducto deferente estão localizadas em posição subcapsular deferencial e abdeferencial e mostram-se providas de válvulas. O trato das artérias testiculares no funículo espermático apresenta como média e desvio padrão 134,6±38,1cm à direita, e 137,0±33,9cm à esquerda, não existindo diferenças estatisticamente significantes ao nível de 5%, quando comparamos a média do segmento da artéria testicular contida no funículo espermático direito em relação ao esquerdo.

Sinais clínicos e ocorrência de anticorpos anti-Chlamydophila abortus em ovinos de São Paulo e Minas Gerais

A Chlamydophila abortus, anteriormente conhecida como Chlamydia psittaci sovovar 1, é uma bactéria Gram negativa, intracelular obrigatória. Esse micro-organismo é frequentemente encontrado em distúrbios reprodutivos em ovinos, bovinos e caprinos, sendo o aborto epizoótico dos bovinos e o aborto enzoótico dos ovinos e caprinos as manifestações mais importantes. Considerando-se o pouco material literário a respeito da clamidofilose no Brasil, a pesquisa teve como objetivo determinar a presença de anticorpos fixadores de complemento anti-Chlamydophila abortus, correlacionando os resultados obtidos com achados no exame clínico e histórico dos animais, além de alterações nos índices zootécnicos, em especial na esfera reprodutiva, tais como alto índice de repetição de cio, número elevado de abortamentos, elevado número de natimortos, entre outros. Foram testadas para prova de fixação do complemento 220 amostras de soro de ovinos, de 26 propriedades, distribuídas em 19 municípios, com relato de manifestação reprodutiva, obtendo-se 19,55% (43/220) de testes positivos para Chlamydophila abortus, com ocorrência de foco constatada de 61,53%. No geral, a titulação de anticorpos encontrada foi baixa, com título não superior a 64. A frequência de manifestação reprodutiva mais observada foi o aborto, representando 65,12% (28/43) do número total de animais soropositivos, seguido de repetição de cio juntamente com nascimento de cordeiro fraco, com frequência de 6,98% (3/ 43) e, por fim, morte neonatal com 4,65% (2/43), sendo que não houve associação significativa entre animais que foram positivos ao teste e a esses fatores.

The complement system of the goat: haemolytic assays and isolation of major proteins

[EN] Background: The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus) and to characterize the major goat complement system proteins. Results: The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions: Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

[EN] Background. Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Los animales domésticos de los habitantes de la Cueva de Villaverde (Fuerteventura)

[ES] More than two hundred and fifty kilogrammes (six hundred pounds) of uncountable osseous fragments of domestic animals from the beginning óf our era have been extracted in the course of archeological excavations carried out in Villaverde. Their inventory and study have proved they belonged to, at least, five hundred goats. fifty sheep, half a dozen swine and two dogs. These animals show affinity with others from the Neolithic and Protohistoric periods of North Africa. Therefore, the goat may be included in the mamber goat group (mambrinus group) and the shepp in "longipes" group, both of them mummified by the Egyptians. Possibly, the swine is similar to the one found in Toukh (Egypt) and the dog may be included in the Saharan Kabyle sheepdog characterized by its small size.

Orígenes de la cabra paleo-canaria de villaverde (Fuerteventura)

[EN] After comparing the features of the group of goats from Villaverde ( osseous remains from the beginning of our era), -La Palma (recently extinct), and - Desertas (descendants of the anciet Canarian goat ), all of them cosidered to be of the same breed, with those of the neolithic and protohistoric goats from North Africa, greater affinity is found with the mamber goat of the predinastic Egypt and the Ancient Egyptian Empire, represented in King Ranusir's tomb (V Dinasty) 4.500 years ago, and affinity is also found with the neolithic goat painted in Amguid (Central Sahara) and the modern Sahel goats, although the paleo - Canarian goat has its own features.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.