Investigations into the phylogeny, genome size, and karyotype of microsporidian Nadeispora canceri were initiated to further characterize the organism. Isolates of N. canceri spores were obtained from both Dungeness (Cancer magisrer) and red rock crabs (Cancer productus). Analysis of the ssu rDNA sequence from spore isolates of the two crab species showed 100% sequence identity among 1,081 nucleotide positions, indicating the same species of microsporidian is infecting both species of crabs. Phylogenetic studies based on the ssu rDNA sequences also showed N. canceri to be most closely related to another crustacean parasite, A meson michaelis. Sequence comparison between the two microsporidian species showed 93% sequence identity (1,001/1081 nucleotide positions). Pulsed field gel electrophoresis was used to estimate the genome size and karyotype of N. canceri isolates obtained from Dungeness and red rock crabs. Resolution of DNA bands on the pulsed field gels revealed both isolates to have a karyotype often chromosome-sized DNA bands. Estimation of the genome size revealed spore isolates from C. magister to have a total genome size of 7.44 Mb and spore isolates from C. productus to have a total genome size of 7.32 Mb. Variations detected in chromosome size culminated in a difference in the genome size between the two isolates. However, the variations in chromosome size were found not to be significant based on the Student's t-test. / Graduation date: 2004

Orientador: Mohamed E. M. Habib / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-19T21:18:37Z (GMT). No. of bitstreams: 1 BrancoJunior_ArmandoCastello_D.pdf: 7897275 bytes, checksum: e274e4921696e8b10fb098edfc48c6c0 (MD5) Previous issue date: 1994 / Resumo: No presente trabalho foram investigados vários aspectos da patologia e epizotiologia de dois agentes biológicos, o microsporídeo Polydispyrenia simulii (Microspora; Duboscquiidae) e o nematódeo Gastromermis viridis cf (Nematoda; Mermithidae), que ocorrem naturalmente no borrachudo Simulium pertinax (Dip.; Simuliidae) na região estudada. Esta espécie de borrachudo apresenta elevada antropofilia, sendo alvo de práticas de controle no sul e sudeste do Brasil. O objetivo principal é a obtenção de dados para a correta avaliação do potencial destes organismos como agentes de controle biológico natural dessa espécie de borrachudo. Quanto aos estudos sobre a microsporidiose em S. pertinax foram feitas investigações histológicas em hospedeiros sadios e doentes, nos diferentes estágios de seu desenvolvimento (larva, pupa e adulto). Verificou-se que em larvas e pupas de S pertinax doentes, o tecido adiposo visceral é o sítio principal de replicação e desenvolvimento do protozoário, havendo também o comprometimento do tecido muscular e do epitélio intestinal no hospedeiro no estágio de larva. No estágio de pupa verificou-se que por processos histolíticos tem-se a debelação da doença e consequente ausência de esporos na fase adulta de S. pertinax. Apesar de não se detectar a presença de esporos nos ovários de fêmeas tanto coletadas no campo como obtidas no laboratório, verificou-se que como consequência da infecção larval, os indivíduos adultos, inclusive machos, apresentam algum comprometimento das gônadas. As fêmeas apresentam uma redução média de 31,2% na fecundidade enquanto que nos machos há uma sensível redução da motilidade dos espermatozóides. Além disso, constatou-se que a manutenção da microsporidiose no campo deve-se muito provavelmente a mecanismos de transmissão horizontal visto que a transmissão vertical não foi confirmada pelas investigações. Quanto aos estudos sobre G. viridis cf em S. pertinax, temos que sua prevalência anual em populações larvais do hospedeiro é baixa, variando de 0,8% a 14,3%. Sua ocorrência parece estar relacionada à vazão do riacho e consequente velocidade de corrente d'água. As investigações histopatológicas revelaram que o tecido adiposo visceral das larvas hospedeiras é o principal alvo da parasitemia, havendo também o comprometimento do tecido muscular e glândulas salivares. Verificou-se ainda que a parasitemia por G. viridis cf. causa redução significativa da resposta comportamental das larvas parasitadas face a qualquer estímulo fisico-químico ou mecânico. Além disso, o retardo no desenvolvimento do hospedeiro larval também foi verificado. Esse fenômeno é conhecido como metatetelia. Os resultados obtidos indicam que o microsporídeo P. simulii tem um significativo papel no controle natural das populações de S. pertinax, uma vez que reduz em quase um terço a fecundidade das fêmeas e promove um menor período de motilidade dos espermatozóides dos machos. Por outro lado, G. viridis cf. apesar de exaurir as reservas da larva hospedeira não apresenta elevada prevalência indicando uma pequena influência na regulação das populações hospedeiras. Acreditamos que alguns estudos ainda são necessários para a correta avaliação do potencial de G. viridis cf. como agente de controle biológico natural de borrachudos. O uso de ambos os agentes, P. simulii e G. viridis cf., nos parece promissor como ferramentas auxiliares para o Manejo Integrado destes incômodos dípteros / Abstract: The aim of the present work is to evaluate the potentiality of Polyd;spyren;a s;mulii (Microspora; Duboscquiidae) and Gastromerm;s v;r;d;s cf (Nematoda; Mermithidae) as two agents for biological control of the blackfly S;mulium pertinax (Diptera; Simuliidae). Pathological and epizootiological aspects of both agents were investigated. Histopathological studies revealed that the visceral fat body of S. pertinax larvae and pupae showed to be the main site of infection for P. s;mulii. The microsporidiosis also atIected the muscular tissue and midgut epithelium of larvae. It was detected that during host pupal development the spores of P. s;mulii were lised, causing the adults to became free of infection. Although no infection was detected in adults originated ITom heavly infected larvae, the number of ovarian folicules of females, as well as the mobility of spermatozoids in males were reduced. It was also detected that vertical transmission does not work on the microsporidiosis maintenance in host populations of S. pertinax. The results also indicated that P. s;mulii has a high influence as a natural control agent of S. pertinax populations. Epizootiological studies reveal that the annual prevalence of the nematode G. v;r;d;s cf in S. pertinax larvae population is low, ranging from 0,8% to 14,3%. Histopathological investigations in infected larvae showed that the visceral fat body was the principal target site of mermithid infection. The muscular tissue and the salivary glands were also atIected. Infected larvae of S. pertinax showed a reduced behavioural response to physical stimulii. Parasitized individuais showed a slower development, known as metatetely. The low prevalence rate of G. v;r;d;s cf in S. pertinax larval populations indicates that its etIects on the blackfly natural control are very small. More studies are necessary for the correct evaluation of G. v;r;d;s cf as biological control agent of S. pertinax. However, both agents may be helpful weapons in Integrated Management Programmes of this medically important insect / Doutorado / Doutor em Ciências Biológicas

No description available.

Thelohania solenopsae Knell, Allen and Hazard, is a microsporidian pathogen that infects the red imported fire ant Solenopsis invicta Buren. This four part study examined the effects that T. solenopsae had on fire ant queens as they matured for their mating flights. For the first study, a total of 878 alates were collected at two timed intervals during a nuptial flight and after to determine if T. solenopsae affected their ability to initiate a mating flight. Infection rates in alates that left the colony early during a flight were 23.75% while alates that did not leave the colony were 66.16%. Two other studies examined whether T. solenopsae affected protein and lipid stores in future queens. Protein stores were not significantly different in infected and uninfected ants, while lipid stores were significantly less in alates that were infected with T. solenopsae (10.69% in infected versus 13.98% in uninfected). The final analysis was done with all of the combined data, which showed that alates infected with T. solenopsae were significantly smaller than uninfected alates.

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

Thelohania solenopsae Knell, Allen and Hazard, is a microsporidian pathogen that infects the red imported fire ant Solenopsis invicta Buren. This four part study examined the effects that T. solenopsae had on fire ant queens as they matured for their mating flights. For the first study, a total of 878 alates were collected at two timed intervals during a nuptial flight and after to determine if T. solenopsae affected their ability to initiate a mating flight. Infection rates in alates that left the colony early during a flight were 23.75% while alates that did not leave the colony were 66.16%. Two other studies examined whether T. solenopsae affected protein and lipid stores in future queens. Protein stores were not significantly different in infected and uninfected ants, while lipid stores were significantly less in alates that were infected with T. solenopsae (10.69% in infected versus 13.98% in uninfected). The final analysis was done with all of the combined data, which showed that alates infected with T. solenopsae were significantly smaller than uninfected alates.

Molecular and in vitro comparisons were performed using two isolates of Encephalitozoon hellem, one from an avian host and one from a human host, and one isolate of Encephalitozoon cuniculi from a rabbit. The molecular comparisons were performed by amplifying and sequencing the gene coding for a zinc metallo-aminopeptidase from cDNA and gDNA obtained from each of the isolates. The E. hellem sequences shared >99 % identity between each other and 70% identity with the E. cuniculi sequences. Conserved HEXXH and GXMEN motifs located within the sequences classify the protein as an aminopeptidase of the M1 family, with at least one zinc atom required for catalytic activity. In vitro growth comparisons of the isolates described above were performed under simulated "mammalian and avian conditions". The models utilized mammalian and avian cell lines and sera at incubation temperatures of 37 °C and 40 °C, respectively. Three separate experiments were performed. E. cuniculi grew best under the mammalian model and significantly better than both E. hellem isolates under this model. The E. hellem isolates were able to infect and replicate under both the mammalian and avian models, which reflects the zoonotic potential of these isolates.

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

Orientador: Gisele Fabrino Machado / Banca:Katia Denise Saraiva Bresciani / Banca: Paulo Ricardo Dell'Armelina Rocha / Resumo: A encefalitozoonose é uma doença zoonótica causada por microsporídios do gênero Encephalitozoon e acomete uma ampla gama de aves, répteis e mamíferos, incluindo os coelhos, principal hospedeiro da espécie Encephalitozoon cuniculi. Esse estudo teve como objetivo investigar a ocorrência da presença do DNA de Encephalitozoon spp. em coelhos de granjas e residências do interior do e estado de São Paulo, Brasil. Foram colhidas 429 amostras por conveniência, que passaram por extração e posterior amplificação de DNA por meio da PCR convencional para o gênero Encephalitozoon. O método utilizado permitiu a detecção do parasito em 11 amostras (2,56%). Há presença do esporo do parasito em algumas destas granjas, mas nenhum coelho doméstico apresentou positividade. A baixa porcentagem de DNA detectado nas amostras analisadas pode indicar uma baixa ocorrência desta infecção nos animais presentes nas granjas analisadas. Esta é a primeira pesquisa sobre o gênero Encephalitozoon em granjas de coelhos e residências no estado de São Paulo. / Abstract: Encephalitozoonosis is a zoonotic disease caused by microsporidia of the genus Encephalitozoon and affects a wide range of birds, reptiles and mammals, including rabbits, the main host of the species Encephalitozoon cuniculi. This study aimed to investigate the occurrence of the DNA of Encephalitozoon spp. in rabbits from farms and residences at cities in state of São Paulo, Brazil. A total of 429 samples of feces were collected for convenience, from rabbits living at farms and residences from the state of São Paulo, Brazil. The DNA was extracted and amplified by conventional PCR for the genus Encephalitozoon. The parasite was detected in 11 samples (2.56%). That showed there's the presence of the parasite spore in some of these farms, but no domestic rabbit showed positivity. The low percentage of DNA detected in the analyzed samples, may indicate a low occurrence of this infection in the animals present in the analyzed farms. This is the first research on the genus Encephalitozoon in farms of rabbits and residences in the state of São Paulo. / Mestre

Microsporidia are an obligate, intracellular fungal pathogen that can cause devastating, disseminating infections in the immunocompromised. Because of the limitations of current medications, microsporidia’s abundant presence in the environment, and an increasing number of at-risk populations, investigation into decreasing microsporidia infectivity is needed. As an intracellular pathogen, microspridial attachment is a vital first step to infection, and if attachment is reduced, previous work shows that infectivity is mitigated. An in silico analysis of Encephalitozoon intestinalis revealed a predicted protein similar in sequence to ADAM (A Disintegrin And Metalloproteinase) proteins. This predicted protein is termed microsporidia ADAM or MADAM. ADAM proteins contain an integrin binding region, which is well known to bind to integrin proteins. Integrins are important receptors for attachment and cell signaling, and several pathogens utilize host integrins as a receptor to aid in attachment during infection. Immunoelectron microscopy demonstrates that MADAM protein is found on the plasma membrane, anchoring disk, and polar tube of E. intestinalis spores. Our hypothesis is that MADAM is involved in the key role of host cell attachment. To this end, a 17 amino acid long section of the MADAM protein was generated that surrounded the integrin binding domain. During spore adherence assays, pretreating host cells with this small peptide protein, significantly decreased E. intestinalis spore attachment to host cells as compared to control samples. These results suggest E. intestinalis cleverly exploits host integrins as a means to bind to host cells before infection.