Molecular and phylogenetic characterization of honey bee viruses, Nosema microsporidia, protozoan parasites, and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana-infecting SBV, and relatively homogenous populations of IAPV and A. meliifera-infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast-encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees

Molecular and phylogenetic characterization of honey bee viruses, Nosema microsporidia, protozoan parasites, and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana-infecting SBV, and relatively homogenous populations of IAPV and A. meliifera-infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast-encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees

Ecological immunology of mosquito-malaria interactions: Of non-natural versus natural model systems and their inferences

There has been a recent shift in the literature on mosquito/Plasmodium interactions with an increasingly large number of theoretical and experimental studies focusing on their population biology and evolutionary processes. Ecological immunology of mosquito-malaria interactions - the study of the mechanisms and function of mosquito immune responses to Plasmodium in their ecological and evolutionary context - is particularly important for our understanding of malaria transmission and how to control it. Indeed, describing the processes that create and maintain variation in mosquito immune responses and parasite virulence in natural populations may be as important to this endeavor as describing the immune responses themselves. For historical reasons, Ecological Immunology still largely relies on studies based on non-natural model systems. There are many reasons why current research should favour studies conducted closer to the field and more realistic experimental systems whenever possible. As a result, a number of researchers have raised concerns over the use of artificial host-parasite associations to generate inferences about population-level processes. Here I discuss and review several lines of evidence that, I believe, best illustrate and summarize the limitations of inferences generated using non-natural model systems

The origin of life: chemical evolution of a metabolic system in a mineral honeycomb?

For the RNA-world hypothesis to be ecologically feasible, selection mechanisms acting on replicator communities need to be invoked and the corresponding scenarios of molecular evolution specified. Complementing our previous models of chemical evolution on mineral surfaces, in which selection was the consequence of the limited mobility of macromolecules attached to the surface, here we offer an alternative realization of prebiotic group-level selection: the physical encapsulation of local replicator communities into the pores of the mineral substrate. Based on cellular automaton simulations we argue that the effect of group selection in a mineral honeycomb could have been efficient enough to keep prebiotic ribozymes of different specificities and replication rates coexistent, and their metabolic cooperation protected from extensive molecular parasitism. We suggest that mutants of the mild parasites persistent in the metabolic system can acquire useful functions such as replicase activity or the production of membrane components, thus opening the way for the evolution of the first autonomous protocells on Earth

Production and characterization of different types of malaria pigment (hemozoin) and their potential use for screening for hemozoin inhibiting drugs

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2012Malarial pigment, hemozoin, appears to modulate the immune system, however it remains controversial if it has stimulatory or inhibitory effects. Biological properties may depend on the type of hemozoin, namely its origin and production method, morphology or size. Natural hemozoin can be obtained from P. falciparum cultures and identical β-hematin can be synthesized from heme. Characterization becomes crucial to confirm size, shape and possible contaminations. Hemozoin production is also a unique antimalarial drug target. There are several heme inhibition assays to screen for drugs with the potential to inhibit hemozoin growth. Yet, these assays are often complex, use highly concentrated, toxic reagents, and not all confirm the end product’s nature. This work aimed to produce and characterize natural hemozoin and synthetic hemozoin, as well as hemozoin-like crystals. Hemozoin-like crystals growth was also investigated to determine if drugs inhibit crystallization, and adapted in an assay format to explore their potential to screen for hemozoin-inhibiting drugs. Hemozoin obtained from different origins was characterized by several methods, including scanning electron microscopy, X-ray diffraction and infrared spectroscopy. Hemozoin-like crystals were grown in broth medium with different drugs to investigate their ability to inhibit crystallization. Despite an overall similarity in morphology and size, differences were detected and characterized between different types of hemozoin, which may be biologically relevant. Although very similar, X-ray diffraction and infrared spectroscopy showed that hemozoin-like crystals were not true hemozoin. However, inhibition results were consistent with previous heme inhibition studies, with chloroquine and amodiaquine presenting the highest potency, followed by other quinolines such as quinine and mefloquine. In conclusion, hemozoin used in immunology should be thoroughly characterized by several complementary methods. Hemozoin-like crystals growth could be successfully used as a novel assay to screen compounds for their hemozoin inhibiting activity, and may be a helpful tool for antimalarial drug screening.A malária, uma das mais importantes doenças infecciosas da actualidade, permanece um problema de saúde pública a nível global, atingindo regiões tropicais e subtropicais em todo o mundo. Esta doença causa grande morbilidade e mortalidade e contribui para o atraso no desenvolvimento social e económico dos países afectados. A doença é causada por parasitas do género Plasmodium, transmitidos por fêmeas de algumas espécies de mosquito Anopheles. Após a fase sexual no mosquito e a fase hepática do ciclo de vida do parasita no Homem, é durante a fase sanguínea que se manifestam os sintomas da doença febril. Factores do hospedeiro e do parasita poderão contribuir para o desenvolvimento de malária severa, levando eventualmente à morte do doente, sendo que a espécie Plasmodium falciparum é responsável pelo maior número de mortes associadas a malária. Apesar dos esforços travados por organizações internacionais no sentido de combater este flagelo, os programas de controlo e erradicação da malária enfrentam vários obstáculos, como a resistência aos insecticidas usados para impregnar redes mosquiteiras, a inexistência de uma vacina eficaz e, principalmente, a grande disseminação de estirpes de parasitas resistentes aos fármacos anti-maláricos actualmente disponíveis. Ainda que sejam pontuais os relatos de resistência ao quinino, utilizado desde o século XVII, este provoca graves efeitos secundários. Independentemente do sucesso de fármacos sintetizados durante o século passado, compostos como a cloroquina, a mefloquina e os antifolatos são hoje inúteis em muitas regiões do mundo. Não obstante a notável eficácia das artemisininas, estes fármacos podem escassear e tornam-se bastante dispendiosos, devendo ser administrados apenas em combinação com outros, de forma a evitar a propagação de resistência. Assim, é urgente descobrir novos fármacos eficazes no combate à malária. O pigmento malárico, também designado hemozoína, é o produto da desintoxicação de moléculas de heme livre, formado por biocristalização no parasita durante a fase intraeritrocítica, após digestão do conteúdo celular do eritrócito infectado. Pensa-se que fármacos anti-maláricos pertencentes à classe das quinolinas interajam com as moléculas de heme, inibindo este processo de biocristalização e resultando na acumulação de heme livre tóxico responsável pela morte do parasita. Alguns autores defendem que as artemisininas actuam de forma semelhante, apesar de este ser assunto de debate. Esta via de desintoxicação de heme pelo parasita é bastante interessante no desenvolvimento de novos fármacos, já que é exclusivo do parasita e parece ser imutável, devido ao facto de não depender de enzimas codificadas pelo parasita passíveis de sofrer alterações por mutações que estivessem na origem de resistência. Existem várias abordagens para investigação e descoberta de fármacos cujo alvo é a formação da hemozoína. Os ensaios in vitro de inibição da cristalização de heme tentam mimetizar a formação de hemozoína recorrendo à síntese de β-hematina, hemozoína sintética considerada idêntica à hemozoína natural, e permitem identificar compostos com potencial para inibir o crescimento do cristal. Todavia, ensaios diferentes variam grandemente em termos dos resultados que produzem e dos próprios métodos e condições utilizados para a sua realização, e nem sempre são suficientemente reprodutíveis nem de simples execução. O presente estudo teve como objectivo produzir e caracterizar hemozoína de diferentes origens, investigar a capacidade de vários fármacos para inibir o crescimento de cristais semelhantes a hemozoína, e o potencial destes cristais para identificar compostos antimaláricos inibidores da formação de hemozoína. A hemozoína sintética foi obtida por catálise acídica, ao passo que a hemozoína nativa foi obtida quer por purificação, quer por simples extracção a partir de culturas de eritrócitos humanos infectados com P. falciparum. Os cristais semelhantes a hemozoína foram cultivados num meio bem definido suplementado com extracto de sangue. Os cristais produzidos foram caracterizados recorrendo a microscopia óptica, de polarização e microscopia electrónica de varrimento, bem como a difracção raio-X e espectroscopia de infravermelho, e investigados em relação à presença de contaminação por heme, Mycoplasma, DNA ou proteína. Os cristais semelhantes a hemozoína foram ainda cultivados em microplaca na presença ou ausência de vários antibióticos e fármacos antimaláricos. Numa primeira abordagem para a caracterização da hemozoína produzida, que visualmente se apresenta preta em suspensão ou pó seco, a microscopia óptica e de polarização permitiram observar algumas diferenças morfológicas, de cor e depolarização entre os cristais obtidos por diferentes métodos. A microscopia electrónica de varrimento revelou-se talvez o método mais adequado para avaliar a morfologia e homogeneidade, evidenciando a forma de paralelepípedo dos cristais de origem nativa e a forma acicular dos cristais de origem sintética e dos cristais semelhantes a hemozoína. Todavia, apenas recorrendo à difracção raio-X e à espectroscopia de infravermelho foi possível distinguir inequivocamente os cristais semelhantes a hemozoína dos cristais de verdadeira hemozoína. Quando purificada, a hemozoína de origens natural e sintética apresenta ausência dos contaminantes investigados, ao passo que a hemozoína nativa extraída, não purificada, se encontra associada a proteínas e ácidos nucleicos. Ainda assim, e apesar de alguns estudos sugerirem um papel de estimulação do sistema imunitário para a hemozoína, a presença de proteínas e DNA pode ser interessante, caso se considere que a hemozoína poderá ser libertada dentro do vacúolo digestivo intacto ou associada a reminiscências desta ou de outras estruturas do parasita. A contaminação com uma quantidade apreciável de heme em cristais semelhantes a hemozoína contribuiu para estabelecer a diferença relativamente à verdadeira hemozoína. A quantificação realizada com o QuantiChrom™ Heme Assay Kit permitiu tirar partido da optimização para amostras de origem biológica e evita o uso de reagentes tóxicos. A obtenção de hemozoína de origem natural foi morosa e dispendiosa, ao passo que a produção de hemozoína sintética e cristais semelhantes a hemozoína foi mais simples e económica. O IFDO (do inglês Ileal Fluid Dependent Organism) foi descrito por Burdon em 1989 após ter sido isolado do fluido de ileostomia de indivíduos com a doença de Crohn. Além de organismo auto-replicativo, pensou-se poder ser uma espécie de cristal formado por constituintes do meio. Semelhante a priões no respeitante à resistência a desinfecção e esterilização, a empresa Steris interessou-se pelo seu potencial como modelo para investigar este tipo de processos, e confirmada a sua natureza hémica, além da birrefringência e cor castanha, pensou-se que poderia ser hemozoína. Apesar da semelhança, uma caracterização mais completa em colaboração com a Steris revelou a diferença dos cristais, e estes foram então designados cristais semelhantes a hemozoína. A avaliação de compostos anti-maláricos e antibióticos como possíveis inibidores do crescimento dos cristais semelhantes a hemozoína permitiu criar um ensaio in vitro para identificar compostos cujo alvo seja a formação de hemozoína. Observando a microplaca após a incubação do cristal na presença de diferentes concentrações de fármaco, foi possível observar visualmente a presença ou ausência de um precipitado escuro no fundo do poço que indicam respectivamente o crescimento ou a inibição da formação dos cristais semelhantes a hemozoína. O presente trabalho reforça a importância da caracterização da hemozoína obtida a partir de diferentes origens. Deve recorrer-se a vários métodos complementares para avaliação da morfologia, tamanho e identidade dos cristais, e testar a presença de moléculas contaminantes que possam apresentar propriedades químicas e biológicas em estudos imunológicos. Contribuir-se-á desta forma para esclarecer resultados ambíguos na investigação do papel da hemozoína na modulação do sistema imunitário do hospedeiro, bem como na avaliação da actividade de antimaláricos por inibição da formação do cristal. Os resultados obtidos sugerem ainda que o ensaio de inibição do crescimento dos cristais semelhantes a hemozoína pode ser usado a par dos ensaios existentes para testar compostos em relação à sua actividade de inibição da formação de hemozoína. O ensaio de inibição do crescimento dos cristais semelhantes a hemozoína é simples de realizar e requer infraestruturas laboratoriais básicas e reagentes relativamente económicos, podendo contribuir assim para a descoberta de novos antimaláricos e para os esforços no controlo da malária

The protozoan nucleus

The nucleus is arguably the defining characteristic of eukaryotes, distinguishing their cell organisation from both bacteria and archaea. Though the evolutionary history of the nucleus remains the subject of debate, its emergence differs from several other eukaryotic organelles in that it appears not to have evolved through symbiosis, but by cell membrane elaboration from an archaeal ancestor. Evolution of the nucleus has been accompanied by elaboration of nuclear structures that are intimately linked with most aspects of nuclear genome function, including chromosome organisation, DNA maintenance, replication and segregation, and gene expression controls. Here we discuss the complexity of the nucleus and its substructures in protozoan eukaryotes, with a particular emphasis on divergent aspects in eukaryotic parasites, which shed light on nuclear function throughout eukaryotes and reveal specialisations that underpin pathogen biology

The impact of conservation translocations on vector-borne parasites : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Palmerston North, New Zealand

Wildlife conservation in New Zealand relies on translocations of endangered species to safe sites. While knowledge of the biology and behaviour of translocated hosts has steadily increased, the role of parasites in wildlife translocations has been largely overlooked. Parasites can affect their host’s survivorship during translocations by causing disease. However, failure to translocate or reintroduce a host specific parasite with its endangered host can contribute to the extinction of the parasite with unforeseen consequences for the future of the host or even the whole ecosystem. The main aims of this study were to establish baseline data on the impact of North Island saddleback translocations on their avian malaria (Plasmodium spp.) parasites as well as gaining further insight into potential vectors in New Zealand. The study was also intended to contribute to the development of recommendations for future parasite screening programmes for native passerine translocations. Saddlebacks and Plasmodium were chosen because of the detailed saddleback translocation history and its known relationship with the parasite. As a result of this study, several Plasmodium lineages previously unrecorded in saddlebacks and New Zealand were identified, for example, the native Kokako01 and one lineage closest related to two lineages from the Americas. Nonetheless, the most frequent lineages found were the cosmopolitan P. elongatum GRW6 and LINN1, and P. vaughani SYAT05, common in birds introduced to New Zealand. This finding suggests that endemic parasites may have already become rare or extinct. In addition, Plasmodium DNA was detected in both native and introduced mosquitoes that may act as vectors. A qPCR assay was developed that was found to be a cost effective and rapid screening tool for the detection of Plasmodium in native birds suffering from acute infection, presenting with clinical symptoms, and in birds that were found dead. . I conclude that future translocations should consider the movement of endemic parasites with their hosts. How this should happen is open for future studies. However, I urge managers to start considering this issue now as New Zealand has already recorded the extinction of one endemic parasite and many more may have already been lost without knowledge

Gastrointestinal parasites of feral cats from Christmas Island

Objective To investigate the gastrointestinal parasites present in feral cats on Christmas Island, with particular interest in the protozoan parasite Toxoplasma gondii. Procedure Faecal and serum samples were collected from 28 and 25 cats respectively that were trapped as part of an ongoing eradication program being run on Christmas Island by the Department of Environment and Conservation. Faecal samples were screened microscopically for helminth and protozoan parasites. Serum samples were screened for antibodies to T gondii using a commercial indirect immunofluorescence assay (IFA) and a latex agglutination test (LAT). Results The most common helminth parasites detected were Toxocara cati (present in 15 of 28 faecal samples), Strongyloides sp (13/28), Aelurostrongylus abstrusus, (7/28), an unidentified capillarid (6/28) and Ancylostoma sp (4/28). Based on serology, T gondii was the most common parasite detected (protozoan or otherwise) with antibodies detected in 24 serum samples by IFA and 23 serum samples by LAT. Conclusion Cats on Christmas Island harbour many of the helminth and protozoan parasites reported from feral cats elsewhere in Australia. The high seroprevalence of T gondii in these cats indicates a high level of exposure to the parasite in this environment

Molecular epidemiology of African sleeping sickness

Human sleeping sickness in Africa, caused by Trypanosoma brucei spp. raises a number of questions. Despite the widespread distribution of the tsetse vectors and animal trypanosomiasis, human disease is only found in discrete foci which periodically give rise to epidemics followed by periods of endemicity A key to unravelling this puzzle is a detailed knowledge of the aetiological agents responsible for different patterns of disease--knowledge that is difficult to achieve using traditional microscopy. The science of molecular epidemiology has developed a range of tools which have enabled us to accurately identify taxonomic groups at all levels (species, subspecies, populations, strains and isolates). Using these tools, we can now investigate the genetic interactions within and between populations of Trypanosoma brucei and gain an understanding of the distinction between human- and nonhuman-infective subspecies. In this review, we discuss the development of these tools, their advantages and disadvantages and describe how they have been used to understand parasite genetic diversity, the origin of epidemics, the role of reservoir hosts and the population structure. Using the specific case of T.b. rhodesiense in Uganda, we illustrate how molecular epidemiology has enabled us to construct a more detailed understanding of the origins, generation and dynamics of sleeping sickness epidemics