Orientador: Regina Maura Bueno Franco / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T17:55:42Z (GMT). No. of bitstreams: 1 Pereira_MirnaAparecida_D.pdf: 2261433 bytes, checksum: 8a2057547b84881643df7c062530dc1e (MD5) Previous issue date: 2008 / Resumo: Cryptosporidium spp. e Giardia spp. são parasitos causadores de gastroenterites destacando se pela elevada incidência de casos, características de resistência aos tratamentos químicos e capacidade de permanência no meio ambiente. O importante papel desses protozoários em vários surtos epidêmicos de veiculação hídrica e alimentar coloca em evidência as hortaliças que, por serem ingeridas cruas, favorecem a aquisição destas parasitoses. No Brasil, são escassos os dados sobre a ocorrência destes protozoários em vegetais como alface e rúcula, alimentos frescos amplamente consumidos pela população. O propósito deste estudo foi investigar a ocorrência destes parasitos em amostras de hortaliças cultivadas na região agrícola de Campinas (SP), e comercializadas pela Central de Abastecimento de Campinas S.A. CEASA, avaliar a água utilizada na irrigação das mesmas, e as condições higiênico sanitárias das Unidades de Produção Agrícola UPAs estudadas (n=15). Para tanto, a presença de oocistos e cistos foi determinada em amostras de alface (Lactuca saliva) e rúcula (Eruca saliva) e parâmetros fisico químicos foram estabelecidos para a água de irrigação destes vegetais, em diferentes épocas do ano. As coletas foram realizadas durante dois anos consecutivos, nos períodos de maior freqüência d<;ls chuvas e de maior ocorrência (esperada) de casos de criptosporidiose em Campinas (devido à acentuada sazonalidade do protozoário no fim do verão e início do outono) e, em períodos que correspondessem a épocas de maior utilização das águas subsuperficiais na irrigação das hortaliças, devido à escassez de chuvas. As diversas amostras de vegetais, após serem lavadas com 100ml de solução contendo Tween 80 (0,01 %), foram filtradas em membranas de ésteres mistos de celulose (47mm de diâmetro e 3/lm de porosidade nominal), seguida de eluição mediante extração mecânica. Após concentração por centrifugação (1050 x g por 10 minutos), os sedimentos resultantes foram examinados mediante a reação de imunotluorescência direta e teste confirmatório das características morfológicas empregando se o corante tluorogênico DAPI (4',6' diamidino 2 phenylindole). Também se utilizou a técnica de centrífugo tlutuação em sulfato de zinco, com o intuito de comparar a eficiência de detecção destes protozoários nos vegetais. Amostras de água de irrigação (2 litros) foram processadas pela mesma metodologia. A presença de cistos de Giardia spp. ocorreu em 4,1% das 120 amostras de hortaliças examinadas, no primeiro e terceiro períodos de coleta, e não foram encontrados na água de irrigação. Cryptosporidium spp. não foi detectado em nenhum dos períodos investigados, tanto em relação às amostras de hortaliças quanto da água de irrigação. A técnica de centrífugo flutuação em sulfato de zinco mostrou se ineficiente, pois não foi revelada a presença de cistos de protozoários e oocistos de Cryptosporidium spp. nas amostras de hortaliças, quando este método foi empregado. A sensibilidade da técnica de filtração em membranas para os vegetais foi de 2,3 % a 63,1 % para oocistos e, para cistos, 1,1 % a 29,3 %. Em amostras de água de irrigação, a sensibilidade desta metodologia variou de 6,0 % a 15 % para Cryptosporidium spp. e, de 23,6 % a 25,8 % para Giardia spp.. Cistos foram encontrados em 6,6 % das amostras de alface, com densidade de 180 a 230 cistos/50g e, 1,6% das amostras de rúcula, com densidade de 180 cistos/50g de folhas de vegetal o que representa elevada importância em Saúde Pública, dada a possibilidade de aquisição de giardiose. O esclarecimento sobre a dispersão destes protozoários no ambiente e o aperfeiçoamento de procedimentos de detecção dos mesmos, quer em amostras hídricas ou alimentares, são vantajosos para o entendimento da epidemiologia destes patógenos / Abstract: Cryptosporidium spp. and Gim-dia spp. are waterborne parasites that cause gastroenteritis. They are known for the high incidence of cases and the chemical resistance to treatments and capacity to stay intact for a prolonged time in environment. Today, the great influence of protozoa in several waterborne and foodborne outbreaks, becomes evident that vegetables eaten raw are dangerous and favor acquisition of intestinal parasitosis. In Brazil, there is little information on transmission of Cryptosporidium spp. and Giardia spp. in fresh vegetables as lettuce and rucola, largely consumed by population. The objective of this study was to investigate the occurrence of Cryptosporidium and Giardia in vegetable samples from agricultural area in Campinas District (SP, Brazil), and commercialized by Central de Abastecimento de Campinas S.A, CEASA, as well to evaluate the water used in vegetable irrigation, and the health and hygiene related conditions of Unidades de Produção Agrícola - UPAs (n = 15). Therefore, the presence of cysts and oocysts was determined in lettuce (Lactuca saliva) and rucola (Eruca saliva) samples and physico chemical parameters were established for irrigation water of vegetables, in different seasons of the year. Collections were made over two consecutive years, in such a way that chqracterized seasons of higher frequency of rains corresponding to months of higher occurrence of cryptosporidiosis cases in Campinas district (due to accentuated seasonality of protozoa in the end of summer and fhe beginning of autumn) and in per~?ds that corresponded to séasons of high peak utilization of subsurface waters in vegetable irrigation due to lack of rain. After washed with 100 ml of Tween 80 (0,01 %) several vegetable samples were filtered through mixed esters of cellulose membranes (4 7mm diameter and 311m nominal porosity), followed by elution by mechanical extraction. After concentration by centrifugation (1050 x g 10 minutes), the resulting sediments were examined by means of direct immunofluorescence reaction and confirmatory morphological test using the fluorogenic stain DAP1 ( 4', 6' diamidino 2 phenylindole), it was also used zinc sulfate centrifugation flotation method to compare detection efficiency of protozoa in vegetables. Irrigation water samples (2L) were processed by the same methodology. The presence of Giardia spp. cysts occurred in 4,1% of 120 vegetable samples, in the first and third collection periods, cysts were not detected in irrigation water. Cryptosporidium spp. was not detected in either investigated seasons Cin relation to vegetable samples and irrigation water) and the presenee of eysts and ooeysts was not detected through zinc sulfate centrifugation flotation method in either vegetable samples. Sensibility of membrane filtration method for vegetables was 2,3 % to 63,1 % oocysts and 1, I % to 29,3 % eysts, respeetively. In irrigation water samples, the sensibility of methodology varied from 6,0 % to 15 % for Cryptosporidium spp. and from 2.,3 % to 25,8 % for Giardia spp.. Cysts were found in 6,6% of lettuce samples with a density of 180 to 230 cysts/50g, and 1,6 % of rueola samples with a density of 180 eysts/50g found on vegetable leaves. Therefore, it has a high importance for public health due to risk faetor for aequisition of giardiasis. The knowledge on protozoa spreading into environment and the improvement of deteetion process in food or water samples are useful for epidemiological understanding of those parasites / Doutorado / Doutor em Parasitologia

Thesis (MSc Food Sc (Food Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Over the last decade, studies on the quality of the water in many of South Africa.s rivers revealed an increase in pollution levels (DWAF, 1996; DWAF, 2000). In agricultural areas of the Western Cape many farmers draw water for irrigation from nearby rivers without knowing what the microbial pollution level is. In order to investigate the current quality of river water in some of the Western Cape.s major rivers and to explore the possibility of pathogen carryover from water onto produce, this research project was initiated. In an exploratory study over a five month period, the microbiological and water chemistry of three selected sites from the upper Berg and two from the Plankenbrug Rivers were assessed. Studied organisms included Salmonella, Staphylococcus, Listeria, endosporeformers, coliforms, Escherichia coli and intestinal Enterococci, while the chemical analysis consisted of pH, alkalinity, conductivity and chemical oxygen demand (COD). Faecal coliform counts ranging from 540 to 1 700 000 cfu.100ml-1 and 490 to 160 000 cfu.100ml-1 were found for the Berg and Plankenbrug Rivers, respectively. The water temperature ranged from 12º to 21ºC with COD values always below 100 mg.L-1. Potential human pathogens such as Salmonella, Staphylococcus, Listeria, endosporeformers, E. coli and intestinal Enterococci were frequently isolated from all five sites that were sampled. These results are of great concern to farmers, fresh produce retailers and consumers alike as the river water is regularly drawn for irrigation of produce that is later consumed raw or after a minimal processing step. From the exploratory study it was concluded that the water from all the sites were not suitable for use in irrigation practices as they regularly exceeded the guidelines for faecal coliforms and E.coli as set out by South African authorities. Irrigation with faecally polluted river water is one way that fruit and vegetables can become contaminated with foodborne pathogens. The risk of disease transmission from potential pathogens present in the irrigation water is influenced by the microbial load present and the numbers carried over to the produce. In this study the carry-over and survival of Escherichia coli on green beans, sugar-snap peas and cocktail tomatoes was assessed under controlled laboratory conditions. The produce was exposed to E.coli under different combinations of exposure times (5, 15 or 30 min), drying times (30 or 120 min) and different inoculum concentration ranges (107, 105, 104, 10³ and 10ª). In all cases a reduction of at least one log value in original inoculum number was found with the 10ª inoculums showing no survivors. Follow-up studies with an exposure time of 60 min and increased drying times of 6 or 12 h. Neither of these parameter changes affected the variation in numbers for the same inoculum or the E.coli survivors. Similar E.coli loads to those detected on the green beans were detected on the sugar-snap peas. The number of survivors on the cocktail tomatoes was much lower than found for the beans and peas. The number of survivors on the cocktail tomatoes was much lower than found for the beans and peas. This was attributed the 'smooth'surface of the tomato skin probably making attachment of the E.coli bacteria difficult. With the exception of the 10ª inoculum range, all E.coli survivors detected on the three types of produce studied exceeded the guideline numbers set for fresh produce. If similar survival patterns are to be found in the environment then results from this study should serve as a warning that the Plankenburg river water is unsafe for use in the irrigation of fresh produce. Some farmers are already treating river water with chlorine prior to irrigation to eliminate the chance of pathogen transfer onto produce. However, this is not a feasible solution for most farmers due to the high cost of implementing a system such as this. / AFRIKAANSE OPSOMMING: Oor die afgelope tien jaar het studies oor die gehalte van die water in baie van Suid-Afrika se riviere 'n toename in besoedelingsvlakke getoon. Baie boere in landbou gebiede van die Wes-Kaap wat watertrek vir besproeiing uit die nabygelee riviere is onbewus van die mikrobiese besoedelingsvlak van die rivier. Hierdie navorsingsprojek is geinisieer ten einde die huidige gehalte van die water in sommige van die Wes-Kaap se riviere te monitor, asook die moontlikheid van patogeen oordrag van die water na vars produkte te ondersoek. Met behulp van 'n verkennende studie oor . vyf maande tydperk, is die mikrobiologiese en water chemie kenmerke van drie geselekteerde moniteringspunte uit die boonste Bergrivier, en twee uit die Plankenburgrivier bemonster. Die bestudeerde organismes het ingesluit: Salmonella, Staphylococcus, Listeria, endosporeformers, fekale kolivorme, Escherichia coli en intestinale Enterococci. Die chemiese parameters het bestaan uit pH, alkaliniteit, geleiding, temperatuur en chemiese suurstof behoeftes (CSB). Fekale kolivorme tellings het gewissel van 540 - 1 700 000 cfu.100ml-1 en 490 tot 160 000 cfu.100ml-1 is gevind vir die Berg- en Plankenburgriviere, onderskeidelik. Die water temperatuur het gewissel van 12 º tot 21ºC met die CSB-waarde altyd onder 100 mg.L-1. Potensiele menslike patogene soos Salmonella, Staphylococcus, Listeria, endosporeformers, E. coli en intestinale Enterococci is dikwels geisoleer uit al vyf die moniteringspunte. Hierdie resultate is van groot belang vir boere, kleinhandelaars en varsprodukte verbruikers omdat die riviere se water gereeld onttrek word vir besproeiing van produkte wat later gebruik word in rou of in minimale geprosesseerde vorm. Vanuit die verkennende en uit die langer termyn studie (12 maande) is die gevolgtrekking gemaak dat die water vanuit die riviere nie geskik is vir gebruik in besproeiingspraktyke waar die riglyne vir fekale kolivorme en E. coli, soos uiteengesit deur die Suid-Afrikaanse owerhede, gereeld oorskry word nie. Besproeiing met fekale besoedelde rivierwater is een manier waarop groente en vrugte besmet kan word met voedsel patogene. Die risiko vir die oordrag van moontlike patogene teenwoordig in die besproeiingswater word beinvloed deur die mikrobiese lading teenwoordig en die getalle wat oorgedra word na die produkte. In hierdie studie is die oordrag en oorlewing van E. coli op groenboontjies, ertjies en tamaties onder beheerde laboratoriumtoestande beoordeel. Die produkte is blootgestel aan E. coli onder verskillende kombinasies van blootstellingstydperke (5, 15 of 30 min), drogingstye (30 of 120 min) en verskillende inokulum konsentrasies (107, 105, 104, 10³ en 10ª). In elke geval is . afname van ten minste een log waarde van die oorspronklike inokulum konsentrasie gevind. Die 10ª inokulum reeks het geen oorlewende selle getoon nie. Opvolg studies is gedoen met 'n blootstellingstyd van 60 min en verlengde drogingstye van 6 of 12 uur. Daar is gevind dat hierdie parameter veranderings die variasie in getalle vir dieselfde inokulum of die oorlewende E. coli nie beinvloed het nie. Soortgelyke E. coli ladings wat op die groenbone aangetref is, is ook op die ertjies aangetref. Die aantal oorlewendes op die tamaties was heelwat laer as die resultate vir die boontjies en ertjies. Dit word toegeskryf aan die "gladde" oppervlak van die tamatie-vel wat waarskynlik die aanhegting van die E. coli selle bemoeilik. Met die uitsondering van die 10ª inokulum reeks, het al die E. coli oorlewende selle gevind op die drie tipes produkte, die riglyn getalle oorskry wat vir vars produkte gestel is. As soortgelyke oorlewingspatrone gevind word in die omgewing, behoort die resultate van hierdie studie as waarskuwing te dien dat die water van die Plankenburgrivier ongeskik is vir die besproeiing van vars produkte. Sommige boere is reeds besig met chloor behandeling van die rivierwater voor besproeiing, om die kans van patogeenoordrag na die produkte uit te skakel. Dit is egter nie 'n haalbare oplossing vir die meeste boere nie as gevolg van die hoe kostes verbonde aan die implementering van' n stelsel soos hierdie.

Thesis (MSc Food Sc (Food Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture in the Western Cape is not only one of the most important economic sectors but also provides many job opportunities. Over the last few years the sustainability of this successful industry has become threatened by the faecal pollution of rivers used to irrigate produce that will be consumed raw or after minimal processing. This situation not only poses an enormous risk to the health of the consumer but also to farmers who stand to lose their export licenses. The purpose of this study was to determine the microbial types and loads in river water, irrigation water and on irrigated produce. A baseline study was done on four sites in two Western Cape rivers. These sites were chosen to allow for the sampling of river water, irrigation water and irrigated produce so as to determine whether a link between the use of contaminated irrigation water and the microbial population found on irrigated produce exists. The physico-chemical analyses used in the study consisted of: pH, alkalinity, water temperature, conductivity and chemical oxygen demand. The microbial monitoring included the aerobic colony counts (ACC) and the enumeration of the total coliforms, faecal coliforms, staphylococci, enterococci, and aerobic and anaerobic sporeformers present in the water samples. The presence or absence of the potential pathogens like E. coli, Listeria and Salmonella, was also determined. During the baseline study faecal coliform counts as high as 160 000 organisms.100 mL-1 were noted in the Plankenburg River, while counts as high as 460 000 organisms.100 mL-1 were found in the Mosselbank River. Apart from this, high numbers of staphylococci and intestinal enterococci were often found, while E. coli, Listeria and Salmonella were present in samples from both of these rivers. Based on the results of the baseline study on the two rivers it was decided to do a more intensive study on the microbial load of the river and irrigation water as well as irrigated produce from the Mosselbank site. Lettuce and cabbages from a commercial farmer’s fields were chosen as the irrigated produce. During the warmer summer months, ACC counts in the river samples peaked at 12 8000 000 cfu.mL-1, while faecal coliform counts of 1 600 000 organisms.100 mL-1 were found. The three potential pathogens (E. coli, Listeria and Salmonella) were present in all the river samples taken during this period. While the counts of indicator bacteria in the irrigation water was often lower, faecal coliform counts as high as 1 600 000 organisms.100 mL-1 and several other potential pathogens were found on the irrigated lettuce and cabbage. This could indicate a possible “build-up” of contamination on the produce with the repeated application of the tainted irrigation water. According to guidelines published by DWAF in 2008, water to be used for irrigation should not contain more than 4 000 organisms.100 mL-1 faecal coliforms if it is used for the irrigation of crops that are to be consumed raw or after a minimal processing step, as this would increase the health risk to the consumer. Guidelines published by the South African Department of Health are even stricter and state that raw vegetables and fruit should not contain more than 200 coliform organisms per gram, while E. coli and L. monocytogenes should be absent in one gram, and Salmonella spp. in 25 grams of the produce, respectively. From the data obtained during this study it was evident that the two rivers monitored regularly contained faecal indicators at levels much higher than those proposed in national and international guidelines for safe irrigation, making them unfit for the irrigation of MPF’s. It could be concluded that the rivers investigated during this study contained high levels of faecal contamination. Since some of the pathogens isolated from the river and irrigation water and the irrigated produce, it suggests a carry-over of microbial contamination from the river water to the irrigated produce. This was, however, only done using the traditional international methods and the presence of specific pathogens should in future be confirmed by means of molecular techniques. / AFRIKAANSE OPSOMMING: Landbou is nie net die een van die belangrikste ekonomiese sektore in die Wes-Kaap nie, maar verskaf ook vele werksgeleenthede. Oor die afgelope paar jaar word die volhoubaarheid van hierdie suksesvolle industrie egter bedreig deur die fekale kontaminasie van riviere wat gebruik word vir die besproeiing van voedsel wat rou of na ‘n minimale prosesserings stap ingeneem word. Hierdie situasie hou nie net ‘n groot gevaar vir die gesondheid van verbruikers in nie, maar ook vir boere wat hul uitvoerlisensies hierdeur kan verloor. Die doel van hierdie studie was om die ladings en tipes mikrobes in rivier water, besproeiingswater en op besproeide produkte vas te stel. ‘n Basiese studie van vier liggings in twee Wes-Kaapse riviere is gedoen. Hierdie liggings is só gekies dat dit moontlik was om die rivier water, besproeiingswater en die besproeide produkte te monitor, en daar sodoende vasgestel kon word of daar ‘n verhouding is tussen die gebruik van gekontamineerde besproeiingswater en die mikrobe populasie wat op die besproeide produkte aanwesig was. Die fisiko-chemiese analises wat gedurende die studie gedoen is, het pH, alkaliniteit, water temperatuur, geleidingsvermoë en die chemiese suurstof vereiste (COD) ingesluit. Die mikrobiese analises het die aërobe kolonie tellings (ACC) en die enumerasie van die totale kolivorme, fekale kolivorme, staphylococci, enterococci en die aërobe en anaërobe spoorvormers ingesluit. Daar is ook vir die aanwesigheid van potensiële patogene soos E. coli, Listeria en Salmonella getoets. Gedurende die basiese studie is fekale kolovorme tellings van so hoog as 160 000 organismes.100mL-1 in die Plankenburg Rivier aangeteken, terwyl tellings van so hoog as 460 000 organismes.100mL-1 in die Mosselbank Rivier gevind is. Hoë tellings stafielokokki en intestinale enterokokki is gereeld genoteer, terwyl E.coli, Listeria en Salmonella uit die waters van beide hierdie riviere geïsoleer is. Gebaseer op hierdie resultate is daar besluit om ‘n meer intensiewe studie van die rivier, besproeiingswater en die besproeide produkte van die Mosselbank Rivier te doen. Blaarslaai en kool van ‘n kommersiële boer se lande is vir hierdie doel gekies. Gedurende die warmer somer maande het die aërobe kolonie tellings in die rivier ‘n piek van 12 800 000 kve.mL-1 bereik, terwyl fekale kolivorme tellings van 1 600 000 organismes.100mL-1 genoteer is. Die drie potensiële patogene (E. coli, Listeria en Salmonella) was aanwesig in al die monsters wat gedurende hierdie tydperk van die rivierwater geneem is. Alhoewel die tellings indikator bakterieë in die besproeiingswater meestal laag was, is tellings fekale kolivorme van so hoog as 1 600 000 kve.100mL-1 en verskeie ander potensiële patogene op die besproeide blaarslaai en kool gevind. Dit kan dui op ‘n moontlike opbou van kontaminasie op die produkte met die herhaalde besproeiing met gekontamineerde besproeiingswater. Volgens die riglyne wat in 2008 deur DWAF gepubliseer is, mag water wat vir die besproeiing van minimaal geprosesseerdevoedsels gebruik word nie meer as 4 000 organismes.100mL-1 bevat nie, aangesien dit die gesondheid van die gebruiker in gevaar mag stel. Die riglyne van die Suid-Afrikaanse Departement van Gesondheid is selfs strenger en beveel aan dat rou vrugte en groente nie meer as 200 kolivorme en geen L. monocytogenes per gram, en geen Salmonella spp. in 25 g van die produk mag bevat nie. Vanuit die data wat tydens hierdie studie ingesamel is, is dit duidelik dat die twee riviere gereeld fekale indikators bevat het teen vlakke baie hoër as wat in die nasionale en internasionale riglyne aanbeveel word. Hierdie water is dus nie geskik vir die besproeiing van minimaal geprosesseerde produkte nie. Die afleiding kan gemaak word dat die riviere wat tydens hierdie studie gemonitor is, hoë vlakke van fekale kontaminasie bevat het. Aangesien sommige van die patogene vanuit beide die rivier- en besproeiingswater, en vanaf die besproeide produkte geïsoleer is, kan dit dui op ‘n moontlike oordrag van mikrobiese kontaminasie vanuit die rivierwater na die besproeide produkte. Tydens hierdie studie is daar egter net van die tradisionele internasionale metodes gebruik gemaak. Vir toekomstige navorsing word dit aanbeveel dat die aanwesigheid van die spesifieke patogene deur die gebruik van molekulêre metodes bevestig word.