According to the World Health Organization, over 200 infectious diseases in humans originate from animals (zoonoses), posing significant threats to human health. Zoonotic agents account for the majority of emerging and re-emerging pathogens. The human-animal interface has been recognised as an important risk factor that facilitates viruses to cross the species barrier and establish infection in humans. This indicates a need to perform surveillance of human populations who are at high risk of zoonotic infection due to their frequent contact with animals, together with the animals to which humans are exposed. The VIZIONS (Vietnam Initiative on Zoonotic Infections) has been conducted to directly respond to that need. The large virus family Picornaviridae include known emerging pathogens that have major impacts on the economies and human and animal health (e.g. foot-and-mouth disease virus, hand foot and mouth disease virus). Some enteroviruses (EVs) and parechoviruses in this family have been shown to be able to infect both humans and animals while a number of new picornaviruses (new EV variants, cosaviruses, cardioviruses, hunniviruses) with unknown pathogenicity and zoonotic potential have been discovered. This thesis, as part of VIZIONS, hopes to address the following gaps in our knowledge of such viruses in six genera (Enterovirus, Parechovirus, Cosavirus, Cardiovirus, Kobuvirus and Hunnivirus) of the family Picornaviridae: 1) The prevalence and genetic diversity of picornaviruses in studied samples 2) The epidemiology and disease association of the identified viruses 3) The overlaps (if any) of picornaviruses circulating in animals and humans 4) Possible animal sources of picornavirus infections in humans In order to do that, over 2,000 faecal samples collected from a wide range of hosts (pigs, rats, bamboo rats, shrews, bats, chickens, ducks, boars, civets, porcupines, monkeys and humans) were screened for picornaviruses by nested PCR and real-time PCR assays. Detection frequencies varied between viruses and sample origins with kobuvirus as the most commonly detected virus, followed by EV, cardiovirus and hunnivirus. Parechovirus and cosavirus were not detected. Comparison of detection frequencies of viruses infecting pigs revealed a disease (diarrhoea) association with porcine kobuvirus (PKV) but not EV infections. However, differences in PKV viral loads between diarrhoeic and non-diarrhoeic pigs were not statistically significant (p = 0.22). In addition, the PKV VP1 sequences from the two pig categories were not phylogenetically distinct. EV VP1 sequences obtained from pigs and boars showed high genetic diversity with four previously known types and nine new types (EV-G8 to -G16). Analyses of complete genome sequences of two new EV types provided evidence for inter-type recombination with a putative breakpoint in the 2A coding region. Similarly, study on samples from monkeys showed endemic infection of EV but no overlap with EV variants in humans was observed. The majority of EV detected in monkeys were novel with evidence for chimeric genomes and putative recombination breakpoints in the 2A region. New criteria for the classification of EV were additionally proposed. Characterization by sequencing of VP4/VP2 and VP1 regions or complete genomes of picornaviruses in rats and bamboo rats also showed relatively high genetic diversity. While these viruses can infect different species of rats, they were again genetically different from viruses detected in the studied human populations. In summary, studies in this thesis provide substantial new information on the prevalence, genetic diversity and disease association of picornaviruses in the studied populations. However, picornaviruses detected from animals were consistently separate from those found in humans, consistent with a relatively limited zoonotic potential of members of the virus family.

Hymenolepis nana is a ubiquitous parasite, found throughout many developing and developed countries. Globally, the prevalence of H. nana is alarmingly high, with estimates of up to 75 million people infected. In Australia, the rates of infection have increased substantially in the last decade, from less than 20% in the early 1990’s to 55 - 60% in these same communities today. Our knowledge of the epidemiology of infection of H. nana is hampered by the confusion surrounding the host specificity and taxonomy of this parasite. The suggestion of the existence of two separate species, Hymenolepis nana von Siebold 1852 and Hymenolepis fraterna Stiles 1906, was first proposed at the beginning of the 20th century. Despite ongoing discussions in the subsequent years it remained unclear, some 90 years later, whether there were two distinct species, that are highly host specific, or whether they were simply the same species present in both rodent and human hosts. The ongoing controversy surrounding the taxonomy of H. nana has not yet been resolved and remains a point of difference between the taxonomic and medical literature. The epidemiology of infection with H. nana in Australian communities is not well understood as the species present in these communities has never been identified with certainty. It is not clear which form of transmission commonly occurs in Australia, whether the H. nana ‘strain/species’ present in the north-west of Western Australia is present in human and rodent hosts, or whether humans harbour their own ‘strain/sub-species’ of Hymenolepis. Furthermore, it is not known whether mice are a potential zoonotic source for transmission of Hymenolepis to human hosts. In this study, 51 human isolates of H. nana were inoculated into highly susceptible laboratory rodent species. However, these failed to develop into adult worms in all instances, including when rodent species were chemically and genetically immunosuppressed.In addition, 24 of these human isolates were also cross-tested in the flour beetle intermediate host, Tribolium confusum. Of these, only one isolate developed to the cysticercoid stage in beetles, yet when inoculated into laboratory rodents, the cysticercoids also failed to develop into adult stage. Since isolates of H. nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular criteria. In the current study, three regions of ribosomal DNA, the small subunit (18S), the first internal transcribed spacer (ITS1) and the intergenic spacer (IGS) were chosen for genetic characterisation of Hymenolepis spp. from rodent and human hosts from a broad geographic range. In addition, a mitochondrial gene, the cytochrome c oxidase subunit 1 (C01) gene and a non-ribosomal nuclear gene, paramyosin, were characterised in a number of Hymenolepis isolates from different hosts. A small PCR fragment of 369 bp, plus a larger fragment of 1223 bp, were sequenced from the 18S gene of reference isolates of H. nana and the rat tapeworm H. diminuta. Minimal sequence variation was found in the two regions of the 18S between these two morphologically distinct, phylogenetically recognised species, H. nana and H. diminuta, and this indicated that the 18S gene was too conserved for further genetic characterisation of isolates of H. nana from different hosts. A large number of human isolates of H. nana (104) were characterised at the ITS1 using PCRrestriction length fragment polymorphism (PCR-RFLP). The profiles obtained were highly variable and often exceeded the original size of the uncut fragment. This was highly suggestive of the existence of ribosomal spacers that, whilst identical in length, were highly variable in sequence. To overcome the problems of the variable PCR-RFLP profiles, further characterisation of the ITS1, by cloning and sequencing 23 isolates of H. nana, was conducted and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences. The sequence differences led to the separation of the isolates into two clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Indeed, the levels of variability were often higher within an individual isolate than between isolates, regardless of whether they were collected from human or mice hosts, which was problematic for phylogenetic analysis. In addition, mixed parasite infections of H. nana and the rodent tapeworm H. microstoma were identified in four humans in this study, which was unexpected and surprising, as there have been no previous reports in the literature documenting humans as definitive hosts for this parasite. Further studies are required, however, to determine if the detection of H. microstoma in humans reflects a genuine, patent infection or an atypical, accidental occurrence. Sequencing of the mitochondrial cytochrome c oxidase 1 gene (C01) in a number of isolates of Hymenolepis nana from rodents and humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse isolates compared to isolates of H. nana from humans. This provided evidence that the mitochondrial C01 gene was useful for identifying genetic divergences in H. nana that were not resolvable using nuclear loci. Despite a morphological identity between isolates of H. nana from rodent and human hosts, the genetic divergence observed between isolates at the mitochondrial locus was highly suggestive that H. nana is a species complex, or “cryptic” species (= morphologically identical yet genetically distinct). In addition, whilst not supported by high bootstrap values, a clustering of the Australian human isolates into one uniform genetic group that was phylogenetically separated from all the mouse isolates was well supported by biological data obtained in this study. To confirm the phylogeny of the C01 tree a small segment of the nuclear gene, paramyosin, was sequenced in a number of isolates from humans and rodents. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. The high sequence conservation of the paramyosin gene characterised in this study did not refute the finding that H. nana may be a cryptic species that is becoming host adapted. It simply did not provide additional data to that already obtained. A DNA fingerprinting tool, PCR-RFLP, of the ribosomal intergenic spacer (IGS), was developed in this study in order to evaluate its usefulness in tracing particular genotypes within a community, thus determining transmission patterns of H. nana between rodent and human hosts. Analysis of the IGS of numerous H. nana isolates by PCR-RFLP identified the presence of copies of the IGS that, whilst similar in length, differed in their sequence. Similar to that observed in the ITS1, the existence of different IGS copies was found in both rodent and human isolates of H. nana, thus the variability was not evidence of the existence of a rodent- or humanspecific genotype. Evaluation of the intergenic spacer (IGS) as a fingerprinting tool suggests that this region of DNA is too variable within individuals and thus, cannot be effectively used for the study of transmission patterns of the tapeworm H. nana between different hosts. In summary, it appears that the life cycle of H. nana that exists in remote communities in the north-west of Western Australia is likely to involve mainly ‘human to human’ transmission. This is supported by both the biological and genetic data obtained for the mitochondrial locus in this study. The role of the intermediate hosts, such as Tribolium spp., in the Hymenolepis life cycle is still unclear, however it would appear that it may be greatly reduced in the transmission of this parasite in remote Australian communities. In the future, it is recommended that further genetic characterisation of faster evolving mitochondrial genes, and/or suitable nuclear genes be characterised in a larger number of isolates of H. nana. The use of techniques which can combine the characterisation of genotype and phenotype, such as proteomics, may also be highly valuable for studies on H. nana from different hosts.

Reproduction de : Thèse d'exercice : Pharmacie : Nantes : 2008. / Bibliogr.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2007. / Titre provenant de l'écran titre. Bibliogr. p. 93-104.

No description available.

Thesis (Ph.D.)--Michigan State University. Dept. of Large Animal Clinical Sciences, 2008. / Title from PDF t.p. (Mar. 27, 2009) Includes bibliographical references. Also issued in print.

Thesis (MSc (Veterinary Tropical Diseases)--University of Pretoria, 2007. / Includes bibliographical references.

Didelphis albiventris, gambá-de-orelha-branca, é um marsupial de hábitos crepusculares e noturnos que se alimenta de frutos, insetos, pequenos répteis e anfíbios, filhotes de aves e pequenos mamíferos. Com a destruição de seu “habitat” natural devido às queimadas e desmatamentos, esses animais têm-se aproximado, cada vez mais, das regiões peridomiciliar e domiciliar, onde procuram abrigo e alimentos. Com o objetivo de conhecer a diversidade de parasitos de D. albiventris e relatar os que apresentam potencial zoonótico, foram examinados 30 exemplares desta espécie, através de necropsia, para coleta de ectoparasitos da superfície externa do corpo e helmintos dos órgãos e conteúdos estomacal e intestinal. Os sifonápteros foram removidos da superfície externa dos animais, conservados em álcool etílico a 70°GL, clarificados em líquido de Nesbitt, desidratados em etanol, diafanizados em creosoto de Faya e montados em lâminas com bálsamo do Canadá para identificação. Os carrapatos foram removidos da superfície externa dos animais, conservados em álcool etílico a 70°GL e identificados ao estereomicroscópio, segundo chaves específicas de Aragão & Fonseca (1961) e Guimarães et al (2001). Os helmintos foram recolhidos com auxílio de estiletes e pinças, clarificados em lactofenol e montados entre lâminas e lamínulas com bálsamo do Canadá para identificação ao microscópio. Do total de animais examinados, 70% estavam infestados com pulgas das espécies Polygenis (Neopolygenis) atopus, Polygenis (Polygenis) rimatus, Polygenis (Polygenis) roberti roberti, Polygenis (Polygenis) sp., Craneopsylla minerva minerva e Ctenocephalides felis felis, todas essas registradas pela primeira vez sobre D. albiventris e, exceto C. felis felis, são também registradas pela primeira vez no estado do Rio Grande do Sul. Carrapatos foram encontrados em 43,33% dos animais examinados, representados pelas espécies Ixodes loricatus, Amblyomma aureolatum e Amblyomma sp, sendo A. aureolatum registrado pela primeira vez parasitando D. albiventris no Brasil. Os helmintos encontrados foram: Filo Nematoda - Capillaria spp. (esôfago, traquéia, faringe e pulmão), Didelphostrongylus hayesi (pulmão), Turgida turgida (estômago), Gnathostoma sp. (estômago e fígado), Travassostrongylus orloffi, Viannaia hamata e Trichuris minuta no intestino delgado e Trichuris didelphis, Cruzia tentaculata e Aspidodera raillieti no intestino grosso; Classe Trematoda – Echinostoma revolutum, Plagiorchis didelphidis, Rhopalias coronatus, R. baculifer, Brachylaema migrans e Didelphodiplostomum variabile, todos no intestino delgado; Classe Cestoda – exemplares da família Diphyllobotriidae, no intestino delgado; e Filo Acanthocephala – Hamanniella microcephala e Centrorhynchus sp., ambos no intestino delgado. Dos helmintos encontrados, os que apresentam potencial zoonótico segundo a literatura são T. turgida, Gnathostoma sp., Capillaria spp., B. migrans, E. revolutum e Família Diphyllobotriidae. Além disso, os sifonápteros e ixodídeos encontrados são potenciais vetores de patógenos que infectam humanos. D. albiventris, portanto, apresenta grande diversidade parasitária, incluindo espécies que podem potencialmente atingir o homem, alertando para a importância destes marsupiais na disseminação de doenças entre animais e humanos.

Made available in DSpace on 2014-06-11T19:35:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-16Bitstream added on 2014-06-13T20:26:19Z : No. of bitstreams: 1 machado_jg_dr_botfmvz.pdf: 2164394 bytes, checksum: 900650ee155d0c853824e1af4b5332b0 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Leishmaniose Visceral é uma zoonose relevante que mais recentemente tem afetado pacientes imunossuprimidos, e tornou-se um sério problema para a saúde pública. O objetivo deste trabalho foi de avaliar a imunopatologia da infecção com Leishmania chagasi em camundongos BALB/c imunossuprimidos com dexametasona até o momento da infecção. Foram realizadas culturas de células esplênicas sem estímulo e com estímulo (Ag de L. chagasi e ConA) para dosagem das citocinas IFN-g, IL-2, IL-4 e IL-10, e determinação das cargas parasitárias do baço e do fígado. Os grupos imunossuprimidos apresentaram os pesos dos baços e porcentagem de CD4 estatisticamente inferiores aos grupos normais no momento da infecção e nos outros momentos de estudo revelaram a produção tanto de citocinas do tipo TH1(IFN-g e IL-2) quanto do tipo TH2 (IL-10). Os grupos infectados se comportaram de maneira semelhante, produzindo citocinas tipo TH1 e TH2, independentemente da imunossupressão. As cargas parasitárias do baço e do fígado destes grupos não apresentaram diferença estatisticamente significante em nenhum dos momentos. Sugere-se que a imunossupressão realizada com a dexametasona não interferiu na infecção com L. chagasi, provavelmente pelo seu efeito reversível. / Visceral Leishmaniasis is a relevant zoonosis infecting immunosuppressed patients, becoming a serious problem to public health in the last years. The objective of this study was to evaluate the immunopathology of Leishmania chagasi infection in BALB/c mice immunosuppressed with dexamethasone until the day of infection. Spleen cells with and without specific stimulus (Ag of L. chagasi and ConA) were cultured for cytokine quantification (IFN-g, IL-2, IL-4 and IL-10) and spleen and liver parasite loads were determined. Immunosuppressed groups showed statistically lower spleen weight and CD4 percentage than normal groups on the day of infection and on the other days of the study, showing the production of TH1(IFN-g and IL-2) and TH2 (IL-10). cytokines. The other infected groups presented the same results, producing TH1 and TH2 cytokines, no matter if they were immunosuppressed and infected. Spleen and liver parasite loads in these groups did not show statistical differences. It was concluded that immunosuppression using dexamethasone did not interfere in L. chagasi infection, probably due its reversible effect.

Didelphis albiventris, gambá-de-orelha-branca, é um marsupial de hábitos crepusculares e noturnos que se alimenta de frutos, insetos, pequenos répteis e anfíbios, filhotes de aves e pequenos mamíferos. Com a destruição de seu “habitat” natural devido às queimadas e desmatamentos, esses animais têm-se aproximado, cada vez mais, das regiões peridomiciliar e domiciliar, onde procuram abrigo e alimentos. Com o objetivo de conhecer a diversidade de parasitos de D. albiventris e relatar os que apresentam potencial zoonótico, foram examinados 30 exemplares desta espécie, através de necropsia, para coleta de ectoparasitos da superfície externa do corpo e helmintos dos órgãos e conteúdos estomacal e intestinal. Os sifonápteros foram removidos da superfície externa dos animais, conservados em álcool etílico a 70°GL, clarificados em líquido de Nesbitt, desidratados em etanol, diafanizados em creosoto de Faya e montados em lâminas com bálsamo do Canadá para identificação. Os carrapatos foram removidos da superfície externa dos animais, conservados em álcool etílico a 70°GL e identificados ao estereomicroscópio, segundo chaves específicas de Aragão & Fonseca (1961) e Guimarães et al (2001). Os helmintos foram recolhidos com auxílio de estiletes e pinças, clarificados em lactofenol e montados entre lâminas e lamínulas com bálsamo do Canadá para identificação ao microscópio. Do total de animais examinados, 70% estavam infestados com pulgas das espécies Polygenis (Neopolygenis) atopus, Polygenis (Polygenis) rimatus, Polygenis (Polygenis) roberti roberti, Polygenis (Polygenis) sp., Craneopsylla minerva minerva e Ctenocephalides felis felis, todas essas registradas pela primeira vez sobre D. albiventris e, exceto C. felis felis, são também registradas pela primeira vez no estado do Rio Grande do Sul. Carrapatos foram encontrados em 43,33% dos animais examinados, representados pelas espécies Ixodes loricatus, Amblyomma aureolatum e Amblyomma sp, sendo A. aureolatum registrado pela primeira vez parasitando D. albiventris no Brasil. Os helmintos encontrados foram: Filo Nematoda - Capillaria spp. (esôfago, traquéia, faringe e pulmão), Didelphostrongylus hayesi (pulmão), Turgida turgida (estômago), Gnathostoma sp. (estômago e fígado), Travassostrongylus orloffi, Viannaia hamata e Trichuris minuta no intestino delgado e Trichuris didelphis, Cruzia tentaculata e Aspidodera raillieti no intestino grosso; Classe Trematoda – Echinostoma revolutum, Plagiorchis didelphidis, Rhopalias coronatus, R. baculifer, Brachylaema migrans e Didelphodiplostomum variabile, todos no intestino delgado; Classe Cestoda – exemplares da família Diphyllobotriidae, no intestino delgado; e Filo Acanthocephala – Hamanniella microcephala e Centrorhynchus sp., ambos no intestino delgado. Dos helmintos encontrados, os que apresentam potencial zoonótico segundo a literatura são T. turgida, Gnathostoma sp., Capillaria spp., B. migrans, E. revolutum e Família Diphyllobotriidae. Além disso, os sifonápteros e ixodídeos encontrados são potenciais vetores de patógenos que infectam humanos. D. albiventris, portanto, apresenta grande diversidade parasitária, incluindo espécies que podem potencialmente atingir o homem, alertando para a importância destes marsupiais na disseminação de doenças entre animais e humanos.