Low Numbers Of Oocysts Research Articles

A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water.

Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures.Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR.Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK.Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water.Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy.Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

Molecular Detection of Cyclospora cayetanensis in Two Main Types of Farm Soil Using Real-Time PCR Assays and Method Modification for Commercial Potting Mix.

Cyclospora cayetanensis is a foodborne protozoan parasite that causes outbreaks of diarrheal illness (cyclosporiasis) with clear seasonality worldwide. In the environment, C. cayetanensis oocysts are very robust, and contact with contaminated soil may serve as an important vehicle in the transmission of this organism, and it is considered a risk factor for this infection. The present study evaluated a flotation concentration method, previously shown to provide the best detection results when compared with DNA isolation directly from soil samples, in two main types of farm soil, silt loam soil and sandy clay loam, as well as in commercial potting mix samples inoculated with different numbers of C. cayetanensis oocysts. The flotation method was able to detect as few as 10 oocysts in 10 g of either type of farm soil without modifications, but needed an extra wash and samples of reduced size for the processing of the commercial potting mix to be able to detect 20 oocysts/5 g. A recently modified real-time PCR method for the detection of C. cayetanensis based on a mitochondrial gene target was also evaluated using selected samples of each type of soil. This comparative study confirmed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is a sensitive method that can detect low numbers of oocysts in different types of soil.

First report of Eimeria myoxi in the garden dormouse (Eliomys quercinus Linnaeus, 1766) from Doñana Natural Area (Andalusia, SW Spain).

This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2±1.2μm and width 17.4±1.1μm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.

Amazonian Anopheles with low numbers of oocysts transmit Plasmodium vivax sporozoites during a blood meal

Anopheles darlingi is the main malarial vector in the Brazilian Amazon region. An. nuneztovari s.l., An. triannulatus s.l., An. evansae, and An. benarrochi s.l. do not have a defined role as malarial vectors, although they have been found to be naturally infected with Plasmodium vivax, and some develop oocysts. In this study, we evaluated the importance of low numbers of oocysts in sporozoite salivary gland invasion and transmission. Field-collected mosquitoes were experimentally infected with P. vivax. The infection rates and oocyst and sporozoite infection intensities were evaluated and compared with those of An. aquasalis. We found the highest number of oocysts in An. darlingi (mean = 39.47) and the lowest in An. nuneztovari s.l. (mean = 2). The highest number of sporozoites was observed in An. darlingi (mean = 610) and lowest in An. benarrochi s.l. (mean = 30). Plasmodium vivax DNA was detected in the saliva of all mosquito species after a blood meal. Regardless of the number of oocysts, all species transmitted sporozoites during blood meals. Considering the abundance of these mosquitoes and transmission of sporozoites, it is logical to assume that An. nuneztovari s.l. and An. triannulatus s.l. are involved in the transmission of P. vivax.

Identification of Three Novel Plasmodium Factors Involved in Ookinete to Oocyst Developmental Transition.

Plasmodium falciparum malaria remains a major cause of global morbidity and mortality, mainly in sub-Saharan Africa. The numbers of new malaria cases and deaths have been stable in the last years despite intense efforts for disease elimination, highlighting the need for new approaches to stop disease transmission. Further understanding of the parasite transmission biology could provide a framework for the development of such approaches. We phenotypically and functionally characterized three novel genes, PIMMS01, PIMMS57, and PIMMS22, using targeted disruption of their orthologs in the rodent parasite Plasmodium berghei. PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. Our results enrich the molecular understanding of the parasite-vector interactions and identify PIMMS01, PIMMS57, and PIMMS22 as new targets of transmission blocking interventions.

Cryptosporidium spp. in Columba livia Gmelin, 1789 (Columbiformes: Columbidae) free-living pigeons from urban areas in Pelotas, Rio Grande do Sul, Brazil.

Cryptosporidium spp. are parasites with zoonotic potential that cause intestinal diseases, generally intense diarrheal, on their hosts, which tend to be immunocompromised. Large populations of pigeons in urban environments can lead to greater human exposure to Cryptosporidium spp., as this bird is considered a potential reservoir and is able to transmit several pathogens. This study aimed in determining the occurrence of Cryptosporidium spp. oocysts in feces of free-living pigeons (Columba livia) found in urban areas in the city of Pelotas, Rio Grande do Sul, south of Brazil. Fecal samples (n = 50) were collected from young and adult pigeons captured in different locations in the urban area and the parasitological diagnosis was performed through Ritchie's modified technique and Kinyoun's technique. Among the 50 samples, 18% (IC95% 9.7-30.8) were positive for Cryptosporidium spp. with a low number of oocysts being detected on fecal smears. Our results confirmed the occurrence of Cryptosporidium spp. oocysts in feces of free-living pigeons from the urban area of the city of Pelotas. This is the first report of Cryptosporidium spp. oocysts in feces of pigeons in south Brazil. This data has epidemiological significance because the oocysts could be from zoonotic species, which consequently shows that humans might be exposed to infection.

Direct Sequencing of Cryptosporidium in Stool Samples for Public Health.

The protozoan parasite Cryptosporidium is an important cause of diarrheal disease (cryptosporidiosis) in humans and animals, with significant morbidity and mortality especially in severely immunocompromised people and in young children in low-resource settings. Due to the sexual life cycle of the parasite, transmission is complex. There are no restrictions on sexual recombination between sub-populations, meaning that large-scale genetic recombination may occur within a host, potentially confounding epidemiological analysis. To clarify the relationships between infections in different hosts, it is first necessary to correctly identify species and genotypes, but these differentiations are not made by standard diagnostic tests and more sophisticated molecular methods have been developed. For instance, multilocus genotyping has been utilized to differentiate isolates within the major human pathogens, Cryptosporidium parvum and Cryptosporidium hominis. This has allowed mixed populations with multiple alleles to be identified: recombination events are considered to be the driving force of increased variation and the emergence of new subtypes. As yet, whole genome sequencing (WGS) is having limited impact on public health investigations, due in part to insufficient numbers of oocysts and purity of DNA derived from clinical samples. Moreover, because public health agencies have not prioritized parasites, validation has not been performed on user-friendly data analysis pipelines suitable for public health practitioners. Nonetheless, since the first whole genome assembly in 2004 there are now numerous genomes of human and animal-derived cryptosporidia publically available, spanning nine species. It has also been demonstrated that WGS from very low numbers of oocysts is possible, through the use of amplification procedures. These data and approaches are providing new insights into host-adapted infectivity, the presence and frequency of multiple sub-populations of Cryptosporidium spp. within single clinical samples, and transmission of infection. Analyses show that although whole genome sequences do indeed contain many alleles, they are invariably dominated by a single highly abundant allele. These insights are helping to better understand population structures within hosts, which will be important to develop novel prevention strategies in the fight against cryptosporidiosis.

Investigation of Cryptosporidium spp. Positivity in Formol-Fixed Stool Samples by Polymerase Chain Reaction Method

Objective: It was aimed to determine Cryptosporidium spp., which is thought to cause digestive system complaints in primary school children, by Modified Acid-Fast (MAF) staining method and Polymerase Chain Reaction (PCR). Method: One hundred sixty four stool samples taken from children were examined under light microscopy with MAF. Positive samples were fixed in 10% formalin and stored at + 4 °C until analysis. These samples were then purified using the Sucrose Gradient method and, DNA isolation was performed using the modified QIAamp DNA Mini Kit (Qiagen) protocol. In order to increase sensitivity of the study, SSU rRNA gene region was amplified by PCR method. Results: Microscopic examination showed that 89 of the 164 stool samples (54,26%) were found to be positive for Cryptosporidium spp.oocysts. Eighty nine samples which were found to be positive by microscopic examination and fixed in %10 formalin were studied by PCR and 74 (45,12%) of them were positive. Conclusions: Cryptosporidiosis, which is known to cause severe infections, especially in immunocompromised individuals and children, is transmitted by fecal-oral route from infected humans and animals, and contaminated water and nutrients. Cryptosporidiosis, which is the result of fecal pollution, causes a great risk for human health even with a low number of oocysts due to the fact that oocysts are resistant to chlorine and they can survive for a long time in the outer environment. It was observed that, primary school children living in area of the study were under risk for cryptosporidiosis.

Immunization of V-line rabbits against intestinal coccidiosis by using attenuated Eimeria oocysts.

The present workaimed to study the immunization of V-line rabbits against coccidiosis by using attenuated Eimeria oocystsbydifferent methods (UVirradiation ,formalin and freezing-thawing ) on Eimeria oocysts and its effect on immune pattern of V-line rabbits. For this aim, Eimeria species (E. exigua,E. flavescens, Emedia, E magna, E. irresidua, E.nagpurensis, E. coecicola and E. intestinalis)were collected from infected and dead rabbits. The collected Eimeria species were sporulated in potassium dichromate 2.5% and divided into four parts ; 1stpart exposed to UV for an hour ,2nd partexposed to six freeze-thaw cycles, 3rd part was diluted with 2.5 ml of formalin 37% and 4th part left without treatment. Fifteen V-line rabbits were classified into 4 groups;G1 was treated by formalin treated Eimeria oocysts,G2 was inoculated by UV irradiated Eimeria oocysts, G3 was inoculated by freezing-thawing oocysts and G4 was inoculated by non-treated Eimeria oocysts. Challenge by non-attenuated Eimeria was carried in 14th day post inoculation.Fecal samples were collected from all groups from 6th to 35thdays, identified and count. Blood samples were collected at days (zero,7th ,14th ,21st,28th and 35th) for determination of cellular and humeral immunity. The obtained results recorded that immunization of rabbits by UV irradiated Eimeria oocysts showed low number of oocysts in feces before and after challenge compared to other groups. The same group showed high level of IgG .It recommended that immunization of rabbits by UV irradiated Eimeria oocysts could protect rabbits against coccidiosis.

Immunization of Lambs Against Coccidiosis by Using Ultraviolet Irradiated Eimeria Oocysts for the First Time

The study was conducted to study the effect of Ultra-violet radiated Eimeria oocysts on immune pattern of lambs. For this purpose, Eimeria sp. (E. crandalis, E. granulosa, E. ovinoidalis, E. parva, E. pallida and E. faurei) were collected from infected sheep and propagated in a lamb for increasing the number of oocysts. The collected Eimeria sp. from the lamb, were sporulated in potassium dichromate 2.5% and divided into 3 parts; the 1st part was exposed to UV radiation for half hour and the 2nd part for one hour and the third left without irradiation. Nine lambs were classified into 3 equal groups; G1 was inoculated by half hour UV irradiated Eimeria oocysts, G2 was inoculated by one-hour UV irradiated Eimeria oocysts and G3 was inoculated by non-irradiated Eimeria oocysts. Fecal samples were collected from all lamb groups from 5th to 21th days, identified and count. Challenge by non-attenuated Eimeria was carried out at 21th day post inoculation. The obtained results revealed that immunization of lambs by using one-hour UV irradiated Eimeria oocysts showed low number of oocysts in feces before and after challenge compared to other groups. The same group also showed high level of γ globulin as well as increasing levels of IgG. It was recommended that immunization of lambs by one-hour UV irradiated Eimeria oocysts could protect lambs against Coccidiosis

Molecular studies on pig cryptosporidiosis in Poland.

Cryptosporidium intestinal parasites have been detected in farmed pigs worldwide. Infections are usually asymptomatic with a low number of oocysts shed in pig feces. This makes the recognition of infection difficult or unsuccessful when microscopic methods are used. The aim of this study was molecular identification of Cryptosporidium species in pig herds raised in Poland with regard to the occurrence of zoonotic species. In total, 166 pig fecal samples were tested. The examined pigs were aged 1 to 20 weeks. Overall, 39 pig farms were monitored for parasite presence. The detection and identification of Cryptosporidium DNA was performed on the basis of PCR-RFLP and nucleotide sequence analysis of the amplified 18 SSU rRNA and COWP gene fragments. Infected animals were housed in 21 (53.8%) of the pig farms monitored. The presence of Cryptosporidum was confirmed in 46 (27.7%) samples of pig feces. Among positive fecal samples, 34 (29.3%) were collected from healthy animals, and 12 (24%) from diarrheic pigs. Most infected animals (42.1%) were 2 to 3 months old. The following parasite species were detected: C. scrofarum, C. suis and C. parvum. Indeed, asymptomatic infections caused by C. scrofarum were observed in the majority of the herds. Mixed infections caused by C. suis and C. scrofarum were not common; however, they were observed in 8.6% of the positive animals. C. parvum DNA was found only in one sample collected from a diarrheic pig. The application of molecular diagnostic tools allowed for detection and identification of Cryptosporidium species in pigs. The sporadic findings of C. parvum are subsequent evidence for the contribution of pigs in the transmission of cryptosporidiosis from animals to humans.

Molecular epidemiology of Cryptosporidium spp. and Giardia spp. in mussels (Mytilus californianus) and California sea lions (Zalophus californianus) from Central California.

Cryptosporidium and Giardia are of public health importance, with recognized transmission through recreational waters. Therefore, both can contaminate marine waters and shellfish, with potential to infect marine mammals in nearshore ecosystems. A 2-year study was conducted to evaluate the presence of Cryptosporidium and Giardia in mussels located at two distinct coastal areas in California, namely, (i) land runoff plume sites and (ii) locations near sea lion haul-out sites, as well as in feces of California sea lions (CSL) (Zalophus californianus) by the use of direct fluorescent antibody (DFA) detection methods and PCR with sequence analysis. In this study, 961 individual mussel hemolymph samples, 54 aliquots of pooled mussel tissue, and 303 CSL fecal samples were screened. Giardia duodenalis assemblages B and D were detected in hemolymph from mussels collected near two land runoff plume sites (Santa Rosa Creek and Carmel River), and assemblages C and D were detected in hemolymph from mussels collected near a sea lion haul-out site (White Rock). These results suggest that mussels are being contaminated by protozoa carried in terrestrial runoff and/or shed in the feces of CSL. Furthermore, low numbers of oocysts and cysts morphologically similar to Cryptosporidium and Giardia, respectively, were detected in CSL fecal samples, suggesting that CSL could be a source and a host of protozoan parasites in coastal environments. The results of this study showed that Cryptosporidium and Giardia spp. from the feces of terrestrial animals and CSL can contaminate mussels and coastal environments.

Large Outbreak ofCryptosporidium hominisInfection Transmitted through the Public Water Supply, Sweden

In November 2010, ≈27,000 (≈45%) inhabitants of Östersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of β-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Phenotypic Dissection of a Plasmodium-Refractory Strain of Malaria Vector Anopheles stephensi: The Reduced Susceptibility to P. berghei and P. yoelii

Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the Plasmodium susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. Anopheles stephensi is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to Plasmodium infection. In the present study, we identified a rodent malaria-refractory strain of A. stephensi mysorensis (Ehime) by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with P. berghei and P. yoelii, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both Plasmodium species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.

The Lesser Egyptian Gerbil (Gerbillus gerbillus) is a suitable host for the long-term propagation of Cryptosporidium andersoni

We describe the course of infection of Cryptosporidium andersoni LI03, originally isolated from cattle, in outbred Gerbillus gerbillus (Lesser Egyptian Gerbil), Meriones unguiculatus (Mongolian gerbil), and Meriones tristrami (Tristram’s jird). While both Meriones spp. partially cleared the infection and shed a low number of oocysts (less than 15,000 oocysts per gram (OPG)), chronic infection with a mean infection intensity reaching 200,000 OPG was observed in G. gerbillus. These data suggest that G. gerbillus can be used as a laboratory model for the long-term maintenance and study of C. andersoni without the need for host immunosuppression.

Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce and a food preparation surface

A proof of concept study was conducted to determine if transparent double-sided adhesive tape could be used to recover and detect [by polymerase chain reaction (PCR) and immunofluorescence microscopy (IFA)] Cryptosporidium parvum oocysts on fresh produce and on a food preparation surface. Oocysts were applied on the surface of ten apples, ten peaches, eight cucumbers, and eight tomatoes within circles drawn with a permanent marker. Approximately 18h later, skin excised from three uncontaminated and three contaminated circles from each piece of produce was subjected to PCR. Pieces of transparent double-sided adhesive tape were lightly pressed onto the surface of three other contaminated circles and examined by PCR. Other pieces of adhesive tape were pressed against the surfaces of three other circles and examined by IFA. At concentrations of 100 and 50 oocysts per circle, every produce item examined by PCR of contaminated excised skin was found positive, and every item examined by adhesive tape subjected to PCR and IFA was found positive, except one. At ten oocysts per circle, every produce item was found positive by PCR of contaminated excised skin, and all apples, cucumbers, and tomatoes were found positive by adhesive tape subjected to IFA. Detection of low numbers of oocysts on peaches by IFA examination of adhesive tape was problematic because trichomes that cover peaches and impart the fuzzy surface partially restrict the tape from reaching some areas where oocysts adhere. Tape combined with IFA was successful in recovering and identifying oocysts from six areas of laminate countertop where the oocysts had been applied and allowed to dry for 30-60min. These are the first findings to demonstrate that adhesive tape can be used to recover and identify a protozoan parasite from fresh produce and from a laminate food preparation surface.

Garlic and hepatic coccidiosis: prophylaxis or treatment?

A study was conducted to investigate the protective and therapeutic effects of crude garlic (Allium sativum) against experimental infection with Eimeria stiedae in rabbits. Forty rabbits were divided into four groups of ten rabbits each: a healthy control group (HC); a challenged-garlic-protected group (CGP) which received a daily dose of 0.5 g/kg body weight (bwt) crude garlic for five successive days before challenge with E. stiedae; a challenged-garlic-treated group (CGT) which was treated with a daily dose of 0.5 g/kg bwt crude garlic for five successive days post-challenge; and an infected control group (IC). The challenge dose was 5 x 10(4) sporulated E. stiedae oocysts per rabbit. Mortality rate, body weight gain, feed conversion ratio and faecal oocyst count were evaluated throughout the experiment. At the end of the experiment, all rabbits were killed and histopathological examination was performed. No mortalities were recorded in the HC and CGP groups, whilst mortality was found to be 20% and 40% in the CGT and IC groups, respectively. CGP rabbits had better body weight gain and lower numbers of oocysts than those in the CGT and IC groups. Hepatic lesions were less severe in the CGP group than in the CGT and IC groups. These results showed that oral administration of crude garlic ameliorated the adverse impacts of hepatic coccidiosis on rabbits when used as a prophylactic, but garlic was less effective as a therapeutic.

A Cryptosporidium hominis outbreak in North-West Wales associated with low oocyst counts in treated drinking water

An outbreak in the autumn of 2005 resulted in 218 confirmed cases of Cryptosporidium hominis. The attack rate (relative risk 4.1, 95%CI 2.8-9.1) was significantly higher in the population supplied by Cwellyn Water Treatment Works (WTW). A case-control study demonstrated a statistically significant association (odds ratio 6.1, 95% CI 1.8-23.8) between drinking unboiled tap water and C. hominis infection. The association remained significant in a logistic regression analysis, with an adjusted odds ratio of 1.30 (95 CI 1.05-1.61) per glass of unboiled tap water consumed per day. This evidence together with environmental and associated microbiological investigations, and the absence of effective treatment to remove Cryptosporidium oocysts at the WTW, led to the conclusion that the outbreak was waterborne. Oocyst counts in final treated water at the WTW and at different points in the distribution system were consistently very low, maximum count in continuous monitoring 0.08 oocysts per 10 litres. Data from continuous monitoring and the epidemic curve is consistent with the hypothesis that low numbers of oocysts of C hominis were present in treated water continuously during the outbreak and these were of sufficient infectivity to cause illness. All surface water derived water supplies present a potential risk to human health and appropriate control measures should be in place to minimise these risks.

Detection of Cryptosporidium species and sources of contamination with Cryptosporidium hominis during a waterborne outbreak in north west Wales

As part of investigations into the cause of a waterborne outbreak of Cryptosporidium hominis infection linked to a mains water supply, surface waters and wastewater treatment plants were tested for Cryptosporidium spp. Oocyst counts in base flow surface water samples ranged from nil to 29 per 10 l. Oocyst counts in effluent from a community wastewater treatment plant were up to 63 fold higher and breakout from one septic tank five logs higher. There were no peak (storm) flow events during the investigation. C. hominis, four named genotypes (cervine, muskrat II, rat, W19) and six new small subunit ribosomal RNA gene sequences were identified. Four of the new sequences were closely related to Cryptosporidium muskrat genotype I, one was closely related to the fox genotype and one to Cryptosporidium canis. C. hominis was found extensively in the catchment, but only at sites contaminated by wastewater, and in the treated water supply to the affected area. All were gp60 subtype IbA10G2, the outbreak subtype. Multiple routes of contamination of the reservoir were identified, resulting in persistent detection of low numbers of oocysts in the final water. This work demonstrates the utility of genotyping Cryptosporidium isolates in environmental samples during outbreak investigations.