Protozoan Contamination Research Articles

Well water sources simultaneous contamination with Cryptosporidium and Acanthamoeba in East-Southeast Asia and Acanthamoeba spp. in biofilms in the Philippines.

Cryptosporidium is the leading agent of waterborne parasitic protozoan outbreaks and is the second leading cause of infant mortality due to diarrhoea worldwide. Acanthamoeba spp. causes Acanthamoeba keratitis (AK) and a life-threatening condition known as granulomatous amoebic encephalitis (GAE). The present study aimed to assess the water quality of an indigenous and a rural community for waterborne parasitic protozoan contamination. Aquatic samples (n = 22) were processed by filtration of 500 mL portion through a 1.2 μm pore size glass microfiber filter and eluted for light microscopy, culture in non-nutrient agar, and PCR analysis. Overall, 36% (8/22) of the investigated aquatic samples were positive for either Cryptosporidium spp. oocysts (13%; 3/22) or Acanthamoeba spp., (36%; 8/22) or both (13%; 3/22). Cryptosporidium spp. oocysts were detected in 27% (3/11) of wet season samples only while Acanthamoeba spp. were detected in 18% (2/11) and 55% (6/11) of wet and dry season samples, respectively. Subsequently, molecular detection for Acanthamoeba species identified A. lenticulata and A. hatchetti with 98-99% BLAST similarity. This is the first report on the simultaneous contamination of Cryptosporidium and Acanthamoeba in well water sources in East-Southeast Asia, the first detection of Acanthamoeba spp. in biofilms in the Philippines, and the longest viability demonstrated for A. lenticulata in two-year-old water samples stored at room temperature.

Sponges (Porifera: Spongillida) as ecological indicators for parasitic protozoans Cryptosporidium and Giardia infective stages in freshwater ecosystems

Sponges filter large volumes of water by actively pumping it through their bodies to acquire nutrients, while Cryptosporidium and Giardia are aquatic ecosystems' primary faecal parasitic pollutants. Five aquatic matrices each of surface water (SW), water sediments (WS), substrate-associated biofilms (SAB), as well as five sponges collected from Lake Buhi were investigated for the presence of parasitic protozoan (oo) cysts. Modified Kinyoun's acid-fast stain (MK) was used to detect Cryptosporidium, while immunofluorescence (IFT) staining was employed to detect both Cryptosporidium and Giardia. Scanning Electron Microscopy (SEM) was used to visualize the skeletal sponge structures and simulated contamination with C. parvum oocysts. Aquatic matrices of WS and SAB demonstrated a 20% (3/15) positivity for Cryptosporidium spp. oocysts with MK and IFT. Overall, the study site prevalence of Cryptosporidium spp. oocysts in aquatic matrices were 60% (3/5). Freshwater sponges demonstrated 100% (5/5) Cryptosporidium and 20% (1/5) Giardia (oo) cysts positivity through IFT. Sponges were ascribed to Radiospongilla cfr. philippinensis (Annandale, 1909) based on diagnostic morphotraits of the siliceous skeleton, i.e., megascleres and gemmuloscleres identified under light microscopy (LM) and SEM. Sponges spiked with Cryptosporidium parvum oocysts simulated parasitic protozoan contamination and demonstrated that oocysts could fit through and be trapped within the sponge aquiferous canals. The presence of Cryptosporidium spp. oocysts in aquatic matrices and Cryptosporidium and Giardia spp. (oo) cysts in Radiospongilla cfr. philippinensis demonstrated the contamination of Porifera with infective stages of both parasites. This study submits a promising perspective on the role of freshwater sponges as ecological indicators, i.e. reservoirs and potential biomonitors for the contamination of infective stages of waterborne parasitic protozoans in aquatic ecosystems.

Global prevalence of intestinal protozoan contamination in vegetables and fruits: A systematic review and meta-analysis

Environmental contamination of vegetables and fruits with intestinal protozoan trophozoites, cysts and oocysts is a means of transmitting parasitic agents of public health importance. The purpose of this systematic review and meta-analysis was to determine the global prevalence of intestinal protozoan parasite contamination in vegetables and fruits. Several databases (Web of Science, PubMed, Scopus, ProQuest and Google Scholar) were searched for literature published up to August 2021. Pooled prevalence was determined using the meta-package in R (version 3.6.1). Out of 90,404 publications, 189 articles (202 datasets) met the inclusion criteria. Among these, 183 investigations documented protozoan contamination in vegetables and 20 in fruits. The pooled prevalence (95% confidence interval) was 20% (16%–24%) for vegetables and 13% (7%–21%) for fruits. The highest pooled prevalence was found in South-East Asian WHO region 37% (6%–76%). The most prevalent protozoan parasite in vegetables was Cryptosporidium spp. (11%, 7%–15%). As well, Entamoeba histolytica was the most common agent found in fruits (9%, 4%–14%). Furthermore, the unwashed samples had the highest pooled prevalence of contamination (22%, 3%–49%). Our data suggest a possible risk of protozoan infection in humans via unwashed vegetables and fruits. Accidental ingestion of protozoa occurs through consumption of contaminated vegetables and fruits that have been improperly washed and prepared under poor sanitation. Using sanitary irrigation water, consuming properly cleaned and cooked vegetables, and practicing good hygiene can all assist to reduce the risk of protozoa infection.

Blue mussel (Mytilus edulis)-A bioindicator of marine water contamination by protozoa: Laboratory and in situ approaches.

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time. In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21days, followed by 21days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa. The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21days). Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters.

A metabarcoding approach for detecting protozoan pathogens in wild oysters from Prince Edward Island, Canada

Food and waterborne protozoan pathogens including Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii are a global concern for human public health. While all three pathogens have been detected in commercial shellfish, there is currently no standard approach for detecting protozoan parasites in shellfish. Common molecular and microscopic methods are limited in the number of pathogens they can simultaneously detect and are often targeted at one or two of these pathogens. Previously, we developed and validated a novel 18S amplicon-based next-generation sequencing assay for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii in shellfish. In this study, we applied the assay for protozoan pathogen detection in wild oysters from Prince Edward Island (PEI). Oysters were harvested from restricted and prohibited areas, classified by the Canadian government according to fecal coliform counts in surrounding waters, and different fractions (whole tissue homogenate and hemolymph) were analyzed. Protozoan DNA was detected using metabarcoding in 28%, of oysters tested (N = 128), and the pathogen read counts in oyster homogenate were considerably higher than those in hemolymph. Protozoan read count thresholds were established for classifying probable oyster contamination with pathogens to account for low levels of background protozoan reads detected in negative controls. Assay results showed protozoan contamination was not associated with harvesting site classifications, suggesting that using fecal indicators for ensuring food safety may be insufficient. Due to the complex matrix, an oyster DNA reduction step may further improve the pathogen detection sensitivity of the assay. Results from this study affirm that novel metabarcoding is a promising screening tool for detection of protozoan pathogens in shellfish.

Quantification of viable protozoan parasites on leafy greens using molecular methods

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

FLOW CYTOMETRIC AND MOLECULAR ANALYSIS OF POSSIBLE PROTOZOAL CONTAMINATION OF DRINKING WATER IN TANTA, EGYPT

Protozoal water contamination is an alarming cause of countless waterborne outbreaks. The mosteminent causal protozoa are Cryptosporidium and Giardia species (spp.) as they can endure aquaticenvironment even with chlorine disinfectants. The currently used traditional techniques cannot permitan easy detection of the waterborne protozoa concerning their count, viability, and pathogenicity. Thepresent work detected protozoal contamination of the drinking water in Egypt with the determinationof their load, viability and potential pathogenicity. Four techniques were compared including conventionalstaining techniques, immunofluorescence (IF) staining, flow cytometry (FC) and molecularstudy. Also, viability was assessed by conventional trypan blue stain and nucleic acid stain. Along ayear, 64 water samples were collected and concentrated from water tanks and tap water of differentdistricts, significant differences (P < 0.001) was between the different techniques in each season regardingthe detection of Giardia cysts and Cryptosporidium oocysts. Number of total positive sampleswas significantly higher in tank water than tap water (P < 0.001) especially at summer. Flow cytometryand nested polymerase chain reaction (nPCR) proved to be much more sensitive than IF assay, andconventional staining techniques. Regarding viability, nucleic acid stain was more sensitive than trypanblue stain (P < 0.001). Also, C. parvum predominate other Cryptosporidium genotypes.

Application of next generation sequencing for detection of protozoan pathogens in shellfish

Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequencing (NGS) for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii spiked into oyster samples. We further developed a bioinformatic pipeline to process and analyze 18S rRNA data for protozoa classification. The ability of the NGS assay to detect protozoa was later compared with conventional PCR. Results demonstrated that background amplification of oyster and other eukaryotic DNA competed with that of protozoa for obtained sequence reads. Sequences of target protozoans were obtained across all spiking levels; however, low numbers of target sequences in negative controls imply that a threshold for true positives must be defined for assay interpretation. While this study focused on three target parasites, the ability of this approach to detect numerous known and potentially unknown protozoan pathogens make it a promising screening tool for monitoring protozoan contamination in food and water.

Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha.

The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7days of exposure at 1000 or 10000 oocysts/mussel/day and 7days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources.

Detection of Cryptosporidium and Giardia in foods of plant origin in North-Western Greece.

Giardia and Cryptosporidium are recognized as leading causes of waterborne and foodborne diarrhoeal disease with worldwide distribution. The study aimed to determine the protozoan contamination of various foods of plant origin. A total of 72 samples from 27 different varieties of fresh vegetables and fruits were collected from supermarkets and open markets in North-Western Greece and were examined using conventional diagnostic methods. Two out of 72 (2.8%) samples were found positive for Cryptosporidium oocysts, while no sample was found to be positive for Giardia cysts. The results show the presence of protozoan contamination in foods of plant origin, which may constitute a potential health hazard.

Use of the bivalve Dreissena polymorpha as a biomonitoring tool to reflect the protozoan load in freshwater bodies

Cryptosporidium parvum, Toxoplasma gondii and Giardia duodenalis are worldwide pathogenic protozoa recognized as major causal agents of waterborne disease outbreaks. To overcome the normative process (ISO 15553/2006) limitations of protozoa detection in aquatic systems, we propose to use the zebra mussel (Dreissena polymorpha), a freshwater bivalve mollusc, as a tool for biomonitoring protozoan contamination. Mussels were exposed to three concentrations of C. parvum oocysts, G. duodenalis cysts or T. gondii oocysts for 21 days followed by 21 days of depuration in clear water. D. polymorpha accumulated protozoa in its tissues and haemolymph. Concerning T. gondii and G. duodenalis, the percentage of protozoa positive mussels reflected the contamination level in water bodies. As for C. parvum detection, oocysts did accumulate in mussel tissues and haemolymph, but in small quantities, and the limit of detection was high (between 50 and 100 oocysts). Low levels of T. gondii (1–5 oocysts/mussel) and G. duodenalis (less than 1 cyst/mussel) were quantified in D. polymorpha tissues. The ability of zebra mussels to reflect contamination by the three protozoa for weeks after the contamination event makes them a good integrative matrix for the biomonitoring of aquatic ecosystems.

Long-term reliable culture of a halophilic diatom, Amphora sp. MUR258, in outdoor raceway ponds

The microalgal industry as a source of high-value products (i.e. β-carotene, astaxanthin) was established over 50 years ago. However, only a very small number of species have been commercialised. There is a need for new species and new products to expand this industry. The objective of this study was to examine the reliability and productivities of long-term outdoor culture of a newly isolated halophilic diatom, Amphora sp. MUR258 (Bacillariophyceae), in raceway ponds in Perth, Western Australia. The Amphora sp. was grown in outdoor raceway ponds as a semi-continuous culture for about 13 months at a culture salinity between 8.6 and 14.9% (w/v) NaCl. The highest cell density (167 × 104 cells mL−1), specific growth rate (0.29 day−1) and biomass and lipid productivities (24 and 6.8 g m−2 day−1, respectively) were achieved in summer. The annual average of biomass (ash-free dry weight) and lipid productivities was 7 and 2.2 g AFDW m−2 day−1, respectively. Minor contamination by a Navicula sp. was seen during winter, but was not a significant problem. No major protozoan contamination was seen. These results indicate the potential of reliable large-scale cultivation of Amphora sp. MUR258 as a potential source of diatom lipid and/or fucoxanthin.

Intestinal protozoa in diarrheic children in an Egyptian rural area: Role of water contamination and other possible risk factors

Background: Poor quality of drinking water has been linked to increased prevalence of diarrheal diseases. Intestinal protozoal infections are potential water borne pathogens. They contribute significantly to the burden of diarrheal diseases. Their transmission dynamics vary considerably among geographic areas.Objective: The present study investigated protozoal infections in diarrheic children and their relation to protozoal contamination of drinking water and other potential risk factors in two Egyptian villages.Material and Methods: Stool samples of 150 diarrheic children and 40 drinking water samples collected from the houses of participating children were examined for enteric protozoa. Data on potential risk factors for protozoal infection were recorded.Results: Protozoa were detected in 62.7% of faecal samples and in 32.5% of water samples. Protozoa were not found in water filtered by domestic filters. Protozoal infection was significantly more common among children who were drinking water in which protozoan contaminants were detected (84.6%) compared to those whose water sources were negative (22.2%). Multivariate logistic regression analysis revealed that only two factors were independently linked to intestinal protozoal infection among participating children; neglecting hand wash before eating (adjusted OR: 7.446, 95% CI: 3.113-17.810) and drinking tap water directly without filtration (adjusted OR: 4.653, 95% CI: 1.82-11.895).Conclusion: The study provides supportive evidence for the role of contaminated water in human infection. Attention should be paid to ensure safe drinking water supply in developing countries. Simple measures such as water filtration coupled with personal hygiene can markedly reduce the risk of infection.

Presence and molecular characterization of Cryptosporidium and Giardia in recreational lake water in Tianjin, China: a preliminary study

Little is known about the occurrence of Cryptosporidium and Giardia in recreational water in China. A total of 52 samples were collected from recreational lakes in Tianjin during a high-occurrence season (June–October) for the waterborne cryptosporidiosis and giardiasis, and the occurrence and genotypes of Cryptosporidium and Giardia were investigated. The results showed that 82.7% (43) and 98.1% (51) of samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively. The mean concentration of parasites was 3.65 oocysts/10 L and 12.58 cysts/10 L, respectively. Molecular characterization revealed that the presence of Cryptosporidium parvum, C. andersoni, C. hominis, C. meleagridis, C. fragile, C. ubiquitum, and Giardia lamblia assemblage A, B and D. The protozoan contamination in the studied lakes may originate from animal feces on ground, which was washed into the lake by stormwater runoff. Nevertheless, there is a potential risk of infection during recreational activities in the lake because the dominant detected protozoan genotypes are common human pathogens. Moreover, microbial indicators analysis does not adequately indicate the protozoan contamination in recreational water. The information from this study will be valuable for future protozoan source tracking, and any further control interventions against Cryptosporidium and/or Giardia infection associated with recreational water.

Assessment of potential sources of protozoan contamination between two avian feeding guilds in a conservation area

Occasional screening of food and water quality available to organisms in protected areas could be beneficial to their successful conservation. This is important for areas receiving regular human visitors and exhibiting activities that may be detrimental to ecosystem health. This study determined the intestinal protozoan species harboured by insectivorous and granivorous birds within the Jos Wildlife Park, Nigeria and whether the two avian feeding guilds are more susceptible to protozoan infection through water or food (grass seeds and insects). Special boxes were used to collect faecal samples from trapped birds. Samples were later analysed in the laboratory for protozoans. Both food and water utilised by the birds in the area were microscopically screened. The composition and abundance of intestinal protozoans between the two feeding guilds did not show significant differences. However, considering parasite species individually, the degree of infection by protozoans such as Giardia lamblia was highly related to the range of infection sources that a feeding guild was exposed to. The composition of parasites observed in the two feeding guilds was strongly linked to both water bodies and avian diet obtainable in the Park. These observations showed that birds in the Park are orally susceptible to infection with protozoans, some of which have been reported to cause health implications in birds. Thus, any measure to curtail environmental contamination by the parasites may improve ecosystem health and survival of avian species in the Park.

Pathogen-reduced platelets for the prevention of bleeding.

Blood for transfusion is collected from donors and then processed and stored as bags of different blood components. One of these components is platelets. Platelets are cells which help the body form clots and prevent bleeding. As for all transfusions, there are risks related to giving platelets to patients, including a small risk of transfusion-transmitted infections. A number of methods are used to minimise the risk of transfusion-transmitted infections, including careful selection of donors and rigorous donor testing. One new method of preventing infection is pathogen reduction by which, through a process of adding chemicals to the donated platelets and exposing them to a wavelength of ultraviolet light, the number of infecting organisms can be reduced. The aim of this review was to assess whether these specially treated, pathogen-reduced platelets, work as effectively as normal platelets when transfused. Specifically, can they stop or prevent bleeding as well as standard platelets, do they produce the same increase in platelet count and does their use affect further transfusion requirements? Also, this review assessed whether pathogen-reduced platelets are as safe as normal platelets - for example, are they associated with any difference in the rate of death following transfusion and are there any side effects associated with the use of these products? Ten randomised controlled trials, involving 1422 patients, were included assessing two different pathogen-reduced platelet transfusion products, Intercept® and Mirasol® platelets, as compared with standard platelets. Nine trials assessed Intercept® platelets and one trial Mirasol® platelets. The 10 trials had different designs with varying methods of outcome assessment and durations of follow-up. Bleeding was assessed as 'any bleeding', 'clinically significant' and 'severe' at short (up to 48 hours) or long (more than seven days) follow-up periods. There was no difference in 'clinically significant' or 'severe bleeding', mortality, transfusion reactions or adverse events between pathogen-reduced and standard platelets. However, there were significantly poorer responses following the transfusion of pathogen-reduced platelets with a requirement for more platelet transfusions. Due to the limitations of the trial sizes and their designs, there is not enough evidence available to be sure that pathogen-reduced platelets work as effectively as standard platelets to prevent bleeding.

Detection and prevalence of protozoan parasites in ready-to-eat packaged salads on sale in Italy

To investigate the prevalence of protozoan contamination by Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Cyclospora cayetanensis, in ‘ready to eat’ (RTE) salads on sale in Italy, 648 packages were purchased from industrial and local brands. Nine individual packages from each brand were collected per month, pooled and subjected to microscopy and molecular analyses. Microscopic examination of 864 slides detected Cryptosporidium spp. but also Blastocystis hominis and Dientamoeba fragilis. Molecular tools identified G. duodenalis assemblage A, Cryptosporidium parvum and Cryptosporidium ubiquitum, T. gondii Type I and C. cayetanensis. B. hominis and D. fragilis were also molecularly confirmed. The overall prevalence of each protozoan species was 0.6% for G. duodenalis, 0.8% for T. gondii, 0.9% for Cryptosporidium spp., and 1.3% for C. cayetanensis, while prevalence for B. hominis was 0.5% and for D. fragilis 0.2%. Microscopy and/or molecular tools revealed that 4.2% of the samples were contaminated by at least one protozoan species, and 0.6% of samples presented contamination by two protozoan species, with a number of oocysts ranging from 62 to 554 per g of vegetable matter for T. gondii, and 46 to 1.580 for C. cayetanensis. This is Europe's first large-scale study on the presence of protozoans in packaged salads, and shows that RTE sanitation processes do not guarantee a product free from protozoans of fecal origin.

Lactose-positive intestinal protozoa in objects under construction soil cover of the Samara Region

Samara Region is one of the largest industrial and agricultural centers of the Russian Federation. It is characterized by a high level of urbanization. Soil degradation, its chemical and bacteriological pollution as well as declining fertility are the result of versatile economic activity in the region. The rate and level of soil pollution are actively monitored however only very scarce data on its sanitary-epidemiological condition are available. At present Samara Region is facing a boost of residential, administrative and industrial constriction which requires sanitary-epidemiological testing of soil at construction sites. The paper deals with the above mentioned problem. It has been determined that the most reliable criteria in estimating the sanitary-epidemiological condition of soil are sanitary-significant microorganisms, a group of coliform bacillus in particular. The causes and effects of urban soil pollution by microbiological contaminants have been analyzed. The role of lactose-positive intestinal protozoa as sanitary-significant microorganisms has been studied. A quantitative evaluation of lactose-positive intestinal protozoa index has been carried out for the soils below residential buildings, administrative buildings, industrial constructions as well as the utility systems area. In particular, it has been measured that the level of soil pollution by lactose positive intestinal protozoa depends on the period they have been in human use. An aggravated level of soil pollution in certain areas can be observed due to uncontrolled littering as well as pet walking or an increasing number of stray animals. Industrialized areas (oil plants) are characterized by the lowest level of lactose positive protozoa contamination which is explained by the fact that those industries use modern technologies which are able to minimize ecological risks. However, such areas are polluted in a different way. Dangerous level of soil contamination by lactose positive protozoa is registered at several large automotive plants.

Zoonotic intestinal protozoan of the wild boars, Sus scrofa, in Persian Gulf's coastal area (Bushehr province), Southwestern Iran.

Aim:Wild boars, Sus scrofa, are potential reservoirs of many zoonotic diseases, and there are a possibility of transmission of the zoonotic diseases from these animals to humans and also domestic animals. This study aimed to evaluate the protozoan contamination of wild boars in the Persian Gulf’s coastal area (Bushehr Province), southwestern Iran.Materials and Methods:A total of 25 crossbred boars were collected during a course of vertebrate pest control in Bushehr province, in 2013. Samples were collected from the gastrointestinal tracts of each boar in 5% formalin, Bouin’s solution, sodium acetate-acetic acid-formalin, and polyvinyl alcohol fixatives. Fixed stool smears examined by trichrome and Ziehl–Neelsen staining.Results:Each of the 25 wild boars was infected with at least one of the intestinal protozoans. The rate of contamination with intestinal protozoan was 64% for Balantidium coli, 76% for Iodamoeba sp., 52% for Entamoeba polecki, 44% for Blastocystis sp. and 8% for Chilomastix sp. No intestinal coccidian was detected in studied boars when the stool samples were evaluated by Ziehl–Neelsen staining method.Conclusion:Findings of this study demonstrated that wild boars in the Persian Gulf coastal area are contaminated by many protozoans, including zoonotic protozoan, which poses a potential risk to locals as well as the domestic animals of the area.

Bioaccumulation of Toxoplasma and Cryptosporidium by the freshwater crustacean Gammarus fossarum: Involvement in biomonitoring surveys and trophic transfer

The protozoa Toxoplasma gondii and Cryptosporidium parvum are public health priorities because their oocysts can persist in recreational, surface, drinking, river, and sea water sources for a long time. To evaluate the capacity of the freshwater crustacean Gammarus fossarum to accumulate T. gondii and C. parvum oocysts, gammarids were exposed to 200, 2000 or 20,000 oocysts per gammarid and per day for 21 days followed by 5 days of depuration. C. parvum DNA was detected by qPCR in G. fossarum in only one out of four pools for the highest concentration and after 14 days of exposure, and T. gondii DNA was detected after 7 days of exposure to the two highest concentrations. Our results document the capacity of G. fossarum to accumulate T. gondii in its tissues proportionally to the ambient concentration; the maximum number of oocysts was detected in gammarid tissues after exposure to 20,000 oocysts per day. Mean values of 3.26 (±3), 21.71 (±15.18), and 17.41 (±10.89) oocysts were detected in gammarids after 7, 14, and 21 days, respectively, and after 5 days of depuration, T. gondii oocysts were still present in gammarid tissues. These results show for the first time that a freshwater crustacean can bioaccumulate T. gondii oocysts, suggesting that G. fossarum is a potential effective bioindicator of protozoan contamination in biomonitoring studies. Moreover, due to its key position in freshwater food webs, G. fossarum could also play a role in the trophic transfer of protozoa.