The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

http://www.marth.com/tkuehn/diss/diss_32_abstract.html / http://www.marth.com/tkuehn/diss/diss_32_abstract.html

Submitted by (edna.saturno@ufrpe.br) on 2016-08-01T16:39:12Z No. of bitstreams: 1 Antonio Tembue.pdf: 785696 bytes, checksum: 3f9e9ef8e3f1459afeea01eeed2f71fa (MD5) / Made available in DSpace on 2016-08-01T16:39:12Z (GMT). No. of bitstreams: 1 Antonio Tembue.pdf: 785696 bytes, checksum: 3f9e9ef8e3f1459afeea01eeed2f71fa (MD5) Previous issue date: 2007-05-18 / Small ruminants production is an important activity specially as a protein source, meat, leather, milk and by-products processing throughout the Brazil. However with the lack of technical support in sheep and goat production systems some parasitic disease has been related including coccidiosis. The goal of this study is describe the epidemiology of some coccidia agents in goats and sheep from Ibimirim County, Pernambuco State, Brazil. A total of 400 animals, being 319 goats and 81 sheep from different farms were studied. First a total of 400 faecal samples were evaluated to identify the eimerian species. Coccidian unsporulated the sporulate oocysts were detected in 95.3% (381/400) and 97.5% (390/400) by using the McMaster technique and aqueous solution of dichromatic (K2Cr2O7) respectively. Eight species of Eimeria were identified: Eimeria arloingi E. ninakohlyakimovae, E. pallida, E. parva, E. intricata, E. ahsata, E. crandallis, E. faurei in goats and E. ahsata, E.crandallis, E. faurei, E. intricata, E. granulosa, E. parva and E. punctata in sheep. The other intestinal coccidia,Cryptosporidium sp oocyst was also identified in 3.7% (3/81), of sheep faeces samples by using Kinyon methodology. On the other hand a serological survey for antibodies to IgG antibodies anti- Toxoplasma gondii and Neospora caninum by Imunofluorescent antibody test were carried out. The results of serology showed the prevalence of 57% (228/400) positive samples to T. gondii, being 58.9% (188/319) and 49.4% (40/81) in goats and sheep respectively. The antibody frequencies according to the age showed that the prevalence was influenced by the age (≤4 years old) of animals in both goats and sheep. The results of imunofluorescent antibody test to Neospora caninum showed 34.3% (137/400) of positive animals, being 26.6% (85/319) in goats and 64.2% (52/81) in sheep. Serologic reactivity was associated with age only in goats (p<0, 01). In conclusion, these results indicate theexposure of small ruminants living in the study area to coccidia of genus Eimeria, Cryptosporidium, Toxoplasma and Neospora. However the frequency of infection of oocysts of Eimeria particularly Eimeria arloingi, E. ninakohlyakimovae em caprinos e Eimeria ahsata e E. crandallis age, and management practices might be consider in order decreasing the effect of these coccidia. In the other hand hygienic management practices should be adopted in these farms in order to prevent the infection of Toxoplasma gondii by the consumption of unpasteurized milk. / A caprinovinocultura é uma importante pecuária atividade, particularmente como fonte de carne, pele, leite e produtos derivados em todo o Brasil. Contudo, com a perda de assistência técnica ao sistema de produção, algumas doenças parasitárias têm sido relatadas, incluindo as coccidioses. Objetivou-se com o presente estudo contribuir no conhecimento da epidemiologia das coccidioses em pequenos ruminantes no Estado de Pernambuco. Um total de 400 animais, sendo 319 caprinos e 81 ovinos,procedentes de diferentes propriedades foram estudados. Inicialmente 400 amostras fecais foram avaliadas pelo método Gordon Whitlock através da técnica de Mc-Master para contagens de oocistos. Foi realizada a esporulação com solução de dicromato de potássio K2Cr2O7 para a identificação das de espécies de gênero Eimeria. Oocistos de coccídios não esporulados e esporulados foram detectados em 95,3% (381/400) e 97, 5% (390/400) pela técnica de McMaster e Solução aquosa de K2Cr2O7. Foram identificadas oito espécies do gênero Eimeria na espécie caprina com as seguintes freqüências relativas por ordem decrescente, Eimeria arloingi; E. ninakohlyakimovae, E. pallida, E. parva, E. intricata, E. ahsata, E. crandallis, E. faurei, enquanto nos ovinos foram encontradas a E. ahsata E.crandallis, E.faurei, E. intricata, E. granulosa, E. parva, e E. punctata. Outro coccídio intestinal, Cryptosporidium spp. oocistos também foi identificado em 3.7% (3/81) das fezes provenientes dos ovinos pelo método de Kinyon. Por outro lado foi realizado um levantamento sorológico de anticorpos IgG anti- Toxoplasma gondii e Neospora caninum através da reação de Imunofluorescencia Indireta. Os resultados revelaram a prevalência de 57% (228/400) de amostras positivas para Toxoplasma gondii, sendo 58,9% (188/319) em caprinos e 49,4% (40/81) em ovinos. A freqüência de anticorpos anti- Toxoplasma gondii foi influenciada com a idade dos animais (≤4 anos) em ambas as espécies animais. O resultado do teste de Imunofluorescencia Indireta para Neospora caninum evidenciou 34,3% (137/400) dos animais foram soropositivoss, sendo 26,6% (85/319) em caprinos e 64,2% (52/81) em ovinos. Com relação à idade, foi observada uma diferença significante somente em caprinos (p<0, 01). De acordo com os resultados obtidos, pode se concluir que os pequenos ruminantes estão expostos à infecção por coccídios de gênero Eimeria, Cryptosporidium, Toxoplasma e Neospora na área estudada. Contudo em função da freqüência de oocistos de gênero Eimeria, observada no presente estudo, particularmente Eimeria arloingi, E. ninakohlyakimovae em caprinos e Eimeria ahsata e E. crandallis em ovinos, medidas higiênico-sanitários devem ser realizadas para minimizar os efeitos produzidos porestes coccídios Não obstante, práticas higiênico-sanitárias devem ser adotadas nas criações para prevenir a infecção de Toxoplasma gondii através do consumo de leite de cabra não pasteurizado.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

http://www.marth.com/tkuehn/diss/diss_32_abstract.html / http://www.marth.com/tkuehn/diss/diss_32_abstract.html

MSc (Microbiology) / Department of Microbiology / Background: Diarrheal diseases constitute an important problem among children but also among HIV positive patients particularly in developing countries such as South Africa. Cryptosporidium infect humans and has been shown to be an important cause of infection among different types of animals. Because of its small size, Cryptosporidium can easily go through the water purification system and can easily become a cause of an epidemic. Previous studies have shown that Cryptosporidium is an important cause of diarrhea in Limpopo Province. However, very few studies have been conducted on the genetic diversity of these organisms in the region. Therefore, the aim of this study was to detect and identify the genetic diversity of Cryptosporidium species from humans and animals in Giyani situated in the northern part of South Africa and Pretoria situated in the central part of the country. Methodology: A total of 560 samples were collected from human and animals and were all screened by microscopy using modified Ziehl-Neelsen staining technique. All the samples were tested by Enzyme-Linked Immunosorbent Assay (ELISA) using the Cryptosporidium II kits from Techlab, Virginia, USA. Positive samples from microscopy and ELISA were examined by different PCR protocols including conventional PCR for amplification of Cryptosporidium oocyst wall protein (COWP) region; Real-time PCR employing SYBR Green detection format for amplification of 18S rRNA region; Real-time PCR employing Hydrolysis probes detection format for amplification of SSU rRNA region; Real-time PCR specific for amplification of C. hominis region and C. parvum region. Positive samples from real-time PCR that gave clear bands on gel electrophoresis were sent for sequencing. The sequences were analysed using Staden package software to edit the nucleotides, Bioedit and MEGA6 software were used to align sequences and draw phylogenetic trees. The SPSS software was used for statistical analysis. xiii Results: The overall prevalence of Cryptosporidium as detected by ELISA method from the samples collected from humans was 41.2% (239/580). The prevalence was higher from the rural area 73.0% (159/218) compared to the urban area 22.1% (80/362) and the difference was statistically significant (χ2 = 145.1; p = 0.0001). Due to the limited amount of samples, only 134 ELISA-positive samples were tested using real-time PCR. Of these samples, 35.8% (48/134) tested positive. Of 48 real-time positive samples 25 were successfully sequenced and two different species (C. hominis and C. muris) were identified. Of all the sequences obtained, one (4.0%) was C. muris and 20 (80%) were C. hominis isolated from rural area, whereas 16.0% (4/25) were also C. hominis isolated from samples obtained from urban area. Cryptosporidium was not associated with diarrhea in the present study. A total of 85 samples were collected from animals (52 from cattle and 33 from goats) and of these 4 (4.7%) were positive by microscopy and ELISA. All these samples were non diarrheal. Conventional PCR also detected a similar number. Of these 4 positive samples, 1 was from a male goat, while the 3 others were obtained from female adult goats. Real-time PCR detected 56.5% (48/85) positive samples. Only 12 of the 85 animal samples were diarrheal and of these 4 were positive for Cryptosporidium. The prevalence of Cryptosporidium infection was higher 68.4% (13/19) in male animals compared to female animals 53.0% (35/66). The prevalence rates in cattle and goats were 55.8% (29/52) and 60.6% (20/33) respectively. Of 48 real-time positive samples from animals, 12 (25.0%) were successfully sequenced and two species (C. parvum and C. andersoni) were identified. Of these 6 were from cattle and the other 6 were from goats. Out of the 12 samples 10 (83%) were C. parvum while 2 (17%) were C. andersoni. Of the two C. andersoni, one was from a goat and one was from a cow. Of the 10 C. parvum, 5 were from goats and 5 were from cattle. xiv In conclusion, microscopy remains the low sensitive tool for the detection of Cryptosporidium while real time PCR appeared to be far much more sensitive by detecting more samples than all the three other methods combined. Closer to the real time PCR was ELISA that detected also more samples compared to conventional PCR and microscopy. The present study identified C. muris from humans’ samples in our area for the first time. However, C. hominis remains the dominant species that infects humans in our area. Cryptosporidium species was mostly found in samples from asymptomatic individuals. In animals, C. parvum was the most commonly isolated organism while C. andersoni was identified in our region for the first time as well and occurred in both goats and cattle. Populations in the affected areas need to be made aware of the infections so that care should be taken to avoid the spread of infection in water sources or in immunocompromised individuals.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below