Enterocytozoon bieneusi y Giardia duodenalis son considerados como los parásitos intestinales más comunes de seres humanos, animales domésticos, silvestres y en cautiverio. Estos patógenos son responsables de causar diarrea en individuos inmunocomprometidos e inmunocompetentes. El presente estudio tuvo por objetivo identificar los genotipos de E. bieneusi y G. duodenalis en muestras de heces de crías de alpaca. Las muestras de heces usadas en el estudio correspondieron a 126 crías de alpaca de 1 a 30 días de edad, de tres regiones geográficas en los andes del Perú (Huancavelica, Cuzco y Puno). Para el diagnostico se amplifico mediante PCR anidado la región espaciador transcrito interno (ITS) del gen ARNr de E. bieneusi, así como el locus triosafosfato isomerasa (TPI) de G. duodenalis. Todas las muestras positivas (E. bieneusi = 65; G. duodenalis = 42) fueron secuenciadas. Sesenta y cinco crías (51.6%) resultaron ser positivas en el PCR para E. bieneusi. Se encontró diez distintos genotipos de E. bieneusi en los aislamientos de crías de alpaca, seis de los aislamientos pertenecen a nuevos genotipos (ALP1, ALP2, ALP3, ALP4, ALP5 y ALP6). Cinco crías (7.7%) fueron positivos para el genotipo P, cuatro crías (6.2%) fueron positivos para el genotipo Tipo IV, dos (3.1%) al genotipo D, una (1.5%) al genotipo Beb6, 48 (73,8%) al genotipo ALP1, y cinco crías fueron positivos para cada genotipo nuevo (ALP2, ALP3, ALP4, ALP5 y ALP6). Así mismo, cuarenta y dos muestras fueron positivas para G. duodenalis. Los ensamblajes A (n = 33) y E (n = 9) fueron detectados en las crías. El ensamblaje A fue el más frecuentemente en crías de Puno y Huancavelica, mientras que el ensamblaje E se encontró en 8 crías en Cuzco y uno de Huancavelica. El papel potencial de E. bieneusi y G. duodenalis en el síndrome diarreico neonatal de la alpaca necesita ser seriamente considerada y evaluada, a pesar de no haber asociación entre la presencia de los parásitos y la presentación de diarrea. Palabras claves: Enterocytozoon bieneusi, Giardia duodenalis, alpaca, diarrea, zoonosis / --- Enterocytozoon bieneusi and Giardia duodenalis are considered the most common intestinal parasites found in humans and domestic, captive and wild animals. These pathogens are responsible for causing diarrhea in immunocompromised and immunocompetent individuals. The aim of the present study was to identify E. bieneusi and G. duodenalis genotypes in fecal samples from alpaca crias. Fecal samples were collected from 126 alpaca crias up to 30 days of age from three geographic regions in the highland of Peru (Huancavelica, Cuzco and Puno). The internal transcribed spacer (ITS) region of the rRNA gene of E. bieneusi, and the amplification of the triosephosphate isomerase (TPI) locus of G. duodenalis, were amplified for a nestedPCR. All positives samples were sequenced. Sixty-five crias (51.6%) were found to be PCR positive for E. bieneusi. Ten distinct genotypes of E. bieneusi were found in alpaca cria isolates, six of all isolates belong to novel genotypes (ALP1, ALP2, ALP3, ALP4, ALP5, and ALP6). Five (7.7%) crias were positive to genotype P, four (6.2%) crias were positive to genotype Type IV, two (3.1%) to genotype D, one (1.5%) to genotype Beb6, 48 (73.8%) to genotype ALP1, and five crias were positive each to new genotype (ALP2, ALP3, ALP4, ALP5, and ALP6). Forty-two samples were positive for G. duodenalis. Assemblages A (n=33) and E (n=9) were detected in crias, Assemblage A was the most frequently found in Puno and Huancavelica, while Assemblage E was found in 8 crias in Cuzco and one from Huancavelica. The potential role of E. bieneusi and G. duodenalis in the neonatal diarrhea syndrome of alpaca needs to be seriously considered and further evaluated, although had not association between the presence of parasites and diarrhea. Key words: Enterocytozoon bieneusi, Giardia duodenalis, alpaca, diarrhea, zoonosis.

Evalúa los parámetros de tiempo, temperatura y concentración de antimicrobianos para el crecimiento de Giardia lamblia en medio de cultivo TYI-S-33 comercial y artesanal. Se llevó a cabo en el Laboratorio de Enteroparásitos de la DEET, del Centro Nacional de Salud Pública del Instituto Nacional de Salud. Se trabajó con la cepa de Giardia intestinalis ATCC 30957 criopreservada a -70°C procedente de un aspirado duodenal de un paciente varón y se reactivó en medio TYI-S-33 comercial, las resiembras se realizaron en medio TYI-S-33 comercial y artesanal, que contenían los antibióticos; penicilina (394 U/ml), estreptomicina (394 μg/ml), gentamicina (50 μg/ml) y amikacina (156 μg/ml), para comparar la carga parasitaria en cada uno de ellos y determinar el mejor medio de cultivo para su desarrollo. Así mismo, se determinó la curva de crecimiento durante 216 horas a tres temperaturas distintas (36°C, 36.5°C y 37°C) determinándose que Giardia lamblia tiene un mejor crecimiento en medio de cultivo TYI-S-33 artesanal a 37°C y una fase log entre las 72 a 120 horas. / Tesis

Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.

Filter-to-waste infrastructure is now commonly incorporated in recently constructed treatment plants and is required by many jurisdictions, including the Nova Scotia Standard for Surface Water Treatment. In the absence of filter-to-waste, operational backwash procedures, such as filter resting, decreased backwash duration, and extended terminal subfluidization wash (ETSW), have shown promise for decreasing ripening time and intensity. Halifax Water’s J.D. Kline Water Supply Plant (JDKWSP) is not equipped with filter-to-waste. Due to the high cost associated with implementing this infrastructure an assessment of filter ripening was performed at this facility to determine if filter-to-waste was needed. The assessment consisted of four studies, including microbial monitoring during filter ripening, testing backwash procedural concepts and backwash water chemistry concepts, and an analysis of the current full-scale procedure.

Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.

This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2??C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25??C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2??C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10??C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20??C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24??C) vs. cold (<2-10??C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.

In South Africa, the assessment of the suitability and acceptability of water for drinking purposes is done according to the South African National Standards (SANS) 241 (2006) which requires that Cryptosporidium and Giardia in drinking water should be less than 1 oocyst/10l and 1 cyst/10l respectively. Although there is a requirement to monitor for these parasitic protozoans, there is lack of uniformity in the monitoring approach. Therefore, the objective of the study was to develop a protocol/methodology that can be applied by drinking water producers to monitor Cryptosporidium and Giardia to ensure that the risk of exposure to these organisms and the risks of non–compliance to guidelines are reduced. Also, to test the feasibility of the protocol on a small system, the drinking water purification plant at the Vaal River Barrage Reservoir that supplies approximately 350 people with drinking water. The protocol for monitoring of Cryptosporidium and Giardia was developed based on monitoring procedures proposed by the US Environmental Protection Agency, the Drinking Water Inspectorate, Australia, New Zealand, and especially on the risk based procedure followed by Northern Ireland with the intention that it will be applicable to all water supply systems irrespective of size and system complexity of the purification works. It is focused on a preventative approach of monitoring Cryptosporidium and Giardia and it consists of ten steps which are: (i) Assessment of the monitoring requirements, (ii) Description and characterization of the source water types (iii) Abstraction of source water (iv) Assessment of the water purification plant (v) Water quality monitoring (vi) Cryptosporidiosis and Giardiasis outbreak (vii) Risk assessment (viii) Sample collection and Laboratory processing (ix) Data evaluation, interpretation and storage (x) Process evaluation and review. As stated, the developed protocol was tested at a small purification plants situated at the dam wall of the Vaal River Barrage catchment, Gauteng Province . From this assessment it was evident that steps of the protocol were easy to follow and the possible risks in the water value chain i.e. from source water to the supply of purified drinking water could be identified. Some of the challenges encountered during the application of the protocol include difficulty in obtaining detailed information regarding the activities around the catchment and information on the prevalence of cryptosporidiosis and giardiasis in the local community or in South Africa in general. From this study, it could be concluded that the source water from the Vaal River Barrage Reservoir was high risk. However, the use of the multi–barrier approach coupled with advanced treatment of UV rendered the water drinking supplied to the local community within the South African Drinking Water Standards for from Cryptosporidium and Giardia of less than 1 oocyst/10l and 1 cyst/10l. The protocol for the monitoring of Cryptosporidium and Giardia could contribute to the protection of drinking water consumers by identifying high risk source waters, identifying areas that can be improved in the water treatment system and also protecting the catchment areas from further faecal pollution. With respect to this outcome, the developed protocol could be used by water utilities as part of their Water Safety Plans to optimize monitoring. Furthermore, this methodology has a potential to contribute to the blue drop certification as it should for part of the Water Safety Plans. / Thesis (M. Environmental Management)--North-West University, Potchefstroom Campus, 2011.

In South Africa, the assessment of the suitability and acceptability of water for drinking purposes is done according to the South African National Standards (SANS) 241 (2006) which requires that Cryptosporidium and Giardia in drinking water should be less than 1 oocyst/10l and 1 cyst/10l respectively. Although there is a requirement to monitor for these parasitic protozoans, there is lack of uniformity in the monitoring approach. Therefore, the objective of the study was to develop a protocol/methodology that can be applied by drinking water producers to monitor Cryptosporidium and Giardia to ensure that the risk of exposure to these organisms and the risks of non–compliance to guidelines are reduced. Also, to test the feasibility of the protocol on a small system, the drinking water purification plant at the Vaal River Barrage Reservoir that supplies approximately 350 people with drinking water. The protocol for monitoring of Cryptosporidium and Giardia was developed based on monitoring procedures proposed by the US Environmental Protection Agency, the Drinking Water Inspectorate, Australia, New Zealand, and especially on the risk based procedure followed by Northern Ireland with the intention that it will be applicable to all water supply systems irrespective of size and system complexity of the purification works. It is focused on a preventative approach of monitoring Cryptosporidium and Giardia and it consists of ten steps which are: (i) Assessment of the monitoring requirements, (ii) Description and characterization of the source water types (iii) Abstraction of source water (iv) Assessment of the water purification plant (v) Water quality monitoring (vi) Cryptosporidiosis and Giardiasis outbreak (vii) Risk assessment (viii) Sample collection and Laboratory processing (ix) Data evaluation, interpretation and storage (x) Process evaluation and review. As stated, the developed protocol was tested at a small purification plants situated at the dam wall of the Vaal River Barrage catchment, Gauteng Province . From this assessment it was evident that steps of the protocol were easy to follow and the possible risks in the water value chain i.e. from source water to the supply of purified drinking water could be identified. Some of the challenges encountered during the application of the protocol include difficulty in obtaining detailed information regarding the activities around the catchment and information on the prevalence of cryptosporidiosis and giardiasis in the local community or in South Africa in general. From this study, it could be concluded that the source water from the Vaal River Barrage Reservoir was high risk. However, the use of the multi–barrier approach coupled with advanced treatment of UV rendered the water drinking supplied to the local community within the South African Drinking Water Standards for from Cryptosporidium and Giardia of less than 1 oocyst/10l and 1 cyst/10l. The protocol for the monitoring of Cryptosporidium and Giardia could contribute to the protection of drinking water consumers by identifying high risk source waters, identifying areas that can be improved in the water treatment system and also protecting the catchment areas from further faecal pollution. With respect to this outcome, the developed protocol could be used by water utilities as part of their Water Safety Plans to optimize monitoring. Furthermore, this methodology has a potential to contribute to the blue drop certification as it should for part of the Water Safety Plans. / Thesis (M. Environmental Management)--North-West University, Potchefstroom Campus, 2011.

Bibliography: leaves 183-228 / x, 228 leaves, [8] leaves of plates : ill. (1 col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989

Thesis (M. Env. Sc.)--University of Adelaide, Mawson Graduate Centre for Environmental Studies, 1999. / Includes bibliographical references (leaves 125-153).