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Babesia bovis has been an important disease agent in the U.S. cattle industry for over a century. Recently, B. bovis-like parasites have been identified in white-tailed deer (WTD; Odocoileus virginianus) in Texas. If the parasites found in the WTD are B. bovis that are able to infect cattle, the disease could re-emerge. Susceptible adult cattle often die from this disease, which would result in severe production losses, as well as a decrease in carcass weights of disease survivors. The B. bovis-like parasite found in WTD was compared to B. bovis from cattle, by ribosomal DNA sequence analysis. Babesia isolated from WTD were found to have 99% identity to B. bovis from GenBank cattle sequences. No cattle samples in this study were found to be positive for B. bovis. On culture of WTD samples, a Babesia parasite could not be visualized based on common morphological features. Trypanosoma cervi has been studied for decades, but all the previous research identified this parasite solely by morphology. Trypanosoma species obtained from different host species was compared by ribosomal DNA sequence analyses. In this study, the Trypanosoma cultured from WTD had the morphological appearance of T. cervi. On sequence analysis, the cattle sequences aligned together with cattle isolates and the WTD sequences aligned closely with elk (Cervus canadensis) sequences, indicating that wild ungulates (WTD and elk) and cattle most likely have separate trypanosome species. On distribution analysis there was a trend in three South Texas counties, where the county with the highest occurrence of Trypanosoma had the lowest occurrence of Babesia; and vice versa. It is possible that Trypanosoma and Babesia blood parasites compete within the mammalian host, but the chi-squared test did not show a significant association between the two parasites in the different counties. On seasonal analysis, the correlation between positive samples and season could not be statistically confirmed, but it appears that Babesia infected animals are found in lowest numbers during hot, dry seasons. It also appears that there is another vector for Trypanosoma in South Texas besides the ked (Lipoptena mazamae) and tabanid fly (Tabanus spp.).

Submitted by ANA PAULA PERES LOPES ROMARIZ (anapaulaperlopes@hotmail.com) on 2018-11-08T20:05:10Z No. of bitstreams: 1 ANA PAULA ROMARIZ Dissertação corrigida 06.05.18 E 04.11.pdf: 2116840 bytes, checksum: b2108011ea2c9d6d8f71c8bf663ffd36 (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2018-11-09T11:06:06Z (GMT) No. of bitstreams: 1 romariz_appl_me_ilha.pdf: 2116840 bytes, checksum: b2108011ea2c9d6d8f71c8bf663ffd36 (MD5) / Made available in DSpace on 2018-11-09T11:06:06Z (GMT). No. of bitstreams: 1 romariz_appl_me_ilha.pdf: 2116840 bytes, checksum: b2108011ea2c9d6d8f71c8bf663ffd36 (MD5) Previous issue date: 2018-03-02 / Tripanosomatídeos são protozoários parasitas flagelados que em ruminantes no Brasil incluem os pertencentes à família Trypanosomatidae, com os gêneros Trypanosoma e Leishmania. Com relação aos búfalos domésticos (Bubalus bubalis), pouco ou nada se conhece sobre estes parasitas. Este estudo teve como objetivo avaliar por meio de métodos parasitológicos (exame direto do parasita no microscópio), sorológicos (RIFI – reação de imunofluorescência indireta) e moleculares (PCR – reação em cadeia pela polimerase e sequenciamento) a presença de tripanosomatídeos em búfalos. Amostras de sangue e suabe conjuntival foram coletadas de 100 búfalos da Fazenda São Francisco, Andradina, SP e de 10 búfalos de Selvíria, MS. Dos 110 búfalos, 60,9% (67/110) apresentaram hemoflagelares tripanossomatídeos nos esfregaços sanguíneos e 90% (99/110) deles apresentaram anticorpos anti-Leishmania pela RIFI. O DNA destes parasitas foi detectado em 29,09% (32/110) dos búfalos pela técnica de PCR, sendo, 26,36% (29/110) na PCR do sangue e 3,6% (4/110) na PCR do suabe conjuntival. Das amostras positivas para PCR, sete foram enviadas para o sequenciamento gênico, e uma delas foi identificada como Leishmania infantum e três como Trypanosoma theileri, ambas as amostras apresentaram 100% de similaridade com as respectivas espécies após comparação pelo GenBank. Este estudo relata o primeiro registro de infecção por L. infantum em B. bubalis na região de Andradina, SP, e a presença de búfalos infectados por espécies de T. theileri, evidenciando a necessidade de mais estudos sobre tripanosomatídeos em búfalos. / Trypanosomatides are flagellate protozoan parasites that in ruminants include those belonging to the family Trypanosomatidae, with the genera Trypanosoma and Leishmania, but with little or nothing information about these parasite infections in domestic water buffalo (Bubalus bubalis). Tthis study was assessed through the parasitological (direct examination of the parasite under microscope), serological (IFAT – immunofluorescence antibody test), and molecular tests (PCR from blood and conjunctival swab and genic sequencing) to detect possible trypanosomatides in buffalo herds from Andradina, SP and Selvíria, MS. Thus, 60.9% (67/110) buffaloes presented trypanosomatid parasites in the blood smears, 90% (99/110) had anti-Leishmania antibodies by IFAT. The DNA of these parasites was detected in 29.09% (32/110) of buffaloes by the PCR technique; 26.36% (29/110) in blood and 3.6% (4/110) in conjunctival swab. Positive PCR samples from seven animals were sent for DNA sequencing, and one of them was identified as Leishmania infantum and three as Trypanosoma theileri, both samples showed 100% of similarity with the respective species after comparison by BenBank. This study reports for the first time the presence of parasite L. infantum in B. bubalis from Andradina, SP, and highlights the presence of buffaloes infected with T. theileri, and indicates the need for more studies on trypanosomatid parasites in buffaloes.

<p> </p><p><em>Trypanosoma theileri</em>, of the subgenus <em>Megatrypanum, </em>a non-pathogenic cosmopolitan blood dwelling parasite of bovine. <em>T. theileri </em>can be cultured at room temperature in several culture media.</p><p>Blood samples were collected from deer's. To see if the blood was infected with trypanosomes it was cultivated in 2 ml sheep blood or cell cultivation medium DMEM with antibiotics.</p><p>Growth was detected by microscopy to see if there were any trypanosomes.</p><p>To determine the species of trypanosomes that was in the deer blood a DNA-preparation was done before a Polymerase Chain Reaction (PCR) could be done. With sequencing the trypanosomes where determined to be<em> Trypanosoma theileri</em>.</p><p>Different tests were made to see in what way the trypanosomes best were caught to the objective slides.</p><p>Forty samples of borrelia positive serum from forty different patients were tested with the fluorescent microscopy. Forty different samples from blood donors were tested the same way.</p><p>Blood samples from 16 different fissiped were taken and to see if they were infected with trypanosomes. Three different PCR's were done on the 16 blood samples.</p><p>A small test on human blood was also performed.</p><p>Protein identification by immunoblot with western blot and silver staining was done.</p><p>With the electron microscopy tests were done in the ordinary way and Critical Dry Point to see if both of the techniques worked.</p><p>Enzyme-Linked Immuno Sorbent Assay (ELISA) test were accomplished on two 96 well plates. The wells on the plates were diluted in different ways before they were processed.</p><p> </p><p> </p>

Trypanosoma theileri, of the subgenus Megatrypanum, a non-pathogenic cosmopolitan blood dwelling parasite of bovine. T. theileri can be cultured at room temperature in several culture media. Blood samples were collected from deer's. To see if the blood was infected with trypanosomes it was cultivated in 2 ml sheep blood or cell cultivation medium DMEM with antibiotics. Growth was detected by microscopy to see if there were any trypanosomes. To determine the species of trypanosomes that was in the deer blood a DNA-preparation was done before a Polymerase Chain Reaction (PCR) could be done. With sequencing the trypanosomes where determined to be Trypanosoma theileri. Different tests were made to see in what way the trypanosomes best were caught to the objective slides. Forty samples of borrelia positive serum from forty different patients were tested with the fluorescent microscopy. Forty different samples from blood donors were tested the same way. Blood samples from 16 different fissiped were taken and to see if they were infected with trypanosomes. Three different PCR's were done on the 16 blood samples. A small test on human blood was also performed. Protein identification by immunoblot with western blot and silver staining was done. With the electron microscopy tests were done in the ordinary way and Critical Dry Point to see if both of the techniques worked. Enzyme-Linked Immuno Sorbent Assay (ELISA) test were accomplished on two 96 well plates. The wells on the plates were diluted in different ways before they were processed.

Orientador: Wilma Aparecida Starcke Buzetti / Resumo: Tripanosomatídeos são protozoários parasitas flagelados que em ruminantes no Brasil incluem os pertencentes à família Trypanosomatidae, com os gêneros Trypanosoma e Leishmania. Com relação aos búfalos domésticos (Bubalus bubalis), pouco ou nada se conhece sobre estes parasitas. Este estudo teve como objetivo avaliar por meio de métodos parasitológicos (exame direto do parasita no microscópio), sorológicos (RIFI – reação de imunofluorescência indireta) e moleculares (PCR – reação em cadeia pela polimerase e sequenciamento) a presença de tripanosomatídeos em búfalos. Amostras de sangue e suabe conjuntival foram coletadas de 100 búfalos da Fazenda São Francisco, Andradina, SP e de 10 búfalos de Selvíria, MS. Dos 110 búfalos, 60,9% (67/110) apresentaram hemoflagelares tripanossomatídeos nos esfregaços sanguíneos e 90% (99/110) deles apresentaram anticorpos anti-Leishmania pela RIFI. O DNA destes parasitas foi detectado em 29,09% (32/110) dos búfalos pela técnica de PCR, sendo, 26,36% (29/110) na PCR do sangue e 3,6% (4/110) na PCR do suabe conjuntival. Das amostras positivas para PCR, sete foram enviadas para o sequenciamento gênico, e uma delas foi identificada como Leishmania infantum e três como Trypanosoma theileri, ambas as amostras apresentaram 100% de similaridade com as respectivas espécies após comparação pelo GenBank. Este estudo relata o primeiro registro de infecção por L. infantum em B. bubalis na região de Andradina, SP, e a presença de búfalos infectados por espéci... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Trypanosomatides are flagellate protozoan parasites that in ruminants include those belonging to the family Trypanosomatidae, with the genera Trypanosoma and Leishmania, but with little or nothing information about these parasite infections in domestic water buffalo (Bubalus bubalis). Tthis study was assessed through the parasitological (direct examination of the parasite under microscope), serological (IFAT – immunofluorescence antibody test), and molecular tests (PCR from blood and conjunctival swab and genic sequencing) to detect possible trypanosomatides in buffalo herds from Andradina, SP and Selvíria, MS. Thus, 60.9% (67/110) buffaloes presented trypanosomatid parasites in the blood smears, 90% (99/110) had anti-Leishmania antibodies by IFAT. The DNA of these parasites was detected in 29.09% (32/110) of buffaloes by the PCR technique; 26.36% (29/110) in blood and 3.6% (4/110) in conjunctival swab. Positive PCR samples from seven animals were sent for DNA sequencing, and one of them was identified as Leishmania infantum and three as Trypanosoma theileri, both samples showed 100% of similarity with the respective species after comparison by BenBank. This study reports for the first time the presence of parasite L. infantum in B. bubalis from Andradina, SP, and highlights the presence of buffaloes infected with T. theileri, and indicates the need for more studies on trypanosomatid parasites in buffaloes. / Mestre

The Trypanosoma theileri group are blood parasites of ungulates. Confirmed vectors of these protists are horseflies or sheep keds for T. melophagium, but these trypanosomes were also detected in deer keds, mosquitoes and phlebotomus. We targeted on the investigation of possibility mosquitoes acting as vectors and additionally we targeted on the prevalence of T. theileri in mosquitoes at selected locations in the Czech Republic due to previous positive detection there. The measured prevalence has reached 6,4 % in mosquitoes, 4 % in deer keds and 16,7 % in sheep keds. The aim of experimental infections was to discover a potential of mosquitoes and phlebotomus acting as vectors of the T. theileri. Phlebotomus perniciosus and three species of mosquitoes Culex pipiens molestus, Culex pipiens quinquefasciatus and Aedes vexans were used for those experiments. Furthermore, isolates of T. theileri and T. melophagium from different sources were used. The divergence among these isolates and among used species of insect was found. The best results were achieved with Ae. aegypti and isolates from mosquitoes, where prevalence had reached 90,8 %. The infected mosquitoes were used for prediuresis research which had shown infection forms of the trypanosomes in the urine liquid. The phylogenetic analyse of T....

Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais. / Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.

Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais. / Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.