A 4-year study of bovine reproductive hormones that are induced by pharmaceuticals and appear as steroid estrogenic pollutants in the resulting slurry, using in vitro and instrumental analytical methods.

The main objective of the research was to study the environmental "price" of the large-scale, milk production from a rarely known perspective, from the mapping of the estrogenic footprint (the amount of oestrus-inducer hormonal products, and the generated endoestrogens) in the resulting slurry in a dairy cow farm. These micropollutants are endocrine-disrupting chemicals (EDCs) and can be dangerous to the normal reproductive functions even at ng/kg concentration. One of them, 17ß-estradiol, has a 20,000 times stronger estrogenic effect than bisphenol-A, a widely known EDC of industrial origin. While most studies on EDCs are short-term and/or laboratory based, this study is longitudinal and field-based. We sampled the slurry pool on a quarterly basis between 2017 and 2020. Our purpose was testing the estrogenic effects using a dual approach. As an effect-based, holistic method, we developed and used the YES (yeast estrogen screen) test employing the genetically modified Saccharomyces cerevisiae BJ3505 strain which contains human estrogenic receptor. For testing exact molecules, UHPLC-FLD was used. Our study points out that slurry contains a growing amount of EDCs with the risk of penetrating into the soil, crops and the food chain. Considering the Green Chemistry concept, the most benign ways to prevent of the pollution of the slurry is choosing appropriate oestrus-inducing veterinary pharmaceuticals (OIVPs) and the separation of the solid and liquid parts with adequate treatment methods. To our knowledge, this is the first paper on the adaptation of the YES test for medicine and slurry samples, extending its applicability. The adapted YES test turned out to be a sensitive, robust and reliable method for testing samples with potential estrogenic effect. Our dual approach was successful in evaluating the estrogenic effect of the slurry samples.

Molecular detection of hemotropic mycoplasmas (hemoplasmas) in domestic cats (Felis catus) in Romania.

BACKGROUND: The hemotropic mycoplasmas (hemoplasmas) of the genus Mycoplasma are recognized as important bacteria that parasitize red blood cells, causing hemolytic anemia in many mammalian species, including cats. No information is available concerning the presence of feline hemoplasma infections in cats in Romania. Thus, the objective of the present study was to provide data on the occurrence and molecular characterization of hemotropic mycoplasmas in client-owned cats in Romania. METHODS: Blood samples from 51 unhealthy cats, originating from Timisoara Municipality, Romania, were screened for the presence of hemoplasmas using conventional polymerase chain reaction (PCR) targeting the 16S rRNA gene and sequencing assays. PCR-positive samples were subsequently analyzed by phylogenetic and population genetic analysis. RESULTS: Molecular analysis revealed 11 (21.6%) positive samples, consisting of 8 (72.7%) Candidatus Mycoplasma haemominutum and 3 (27.3%) Mycoplasma haemofelis confirmed positives. Candidatus Mycoplasma turicensis was not detected, and no co-infections were registered. No significant associations (p > 0.05) were found between the hemoplasma infection status and age, gender, breed, presence of ectoparasites, feline leukemia virus/feline immunodeficiency virus positivity of cats, or the sampling season. However, outdoor access was positively associated (p = 0.049) with infection and could be considered a risk factor (OR = 4.1) in acquiring feline hemotropic mycoplasmas. Phylogenetic analysis revealed that our sequences clustered with those selected from the GenBank database in two distinct clades. The registered population genetic indices were strongly supportive of the great variance in sequences between the recorded Mycoplasma species. CONCLUSIONS: The findings support the occurrence of feline hemoplasma infections in previously uninvestigated territories of Europe, providing useful information for small animal practitioners. To our knowledge, the present survey is the first reported molecular evidence of feline hemoplasma infections in Romania.

Sarcocystis spp. in Romanian Slaughtered Cattle: Molecular Characterization and Epidemiological Significance of the Findings.

Species of the genus Sarcocystis are recognized as protozoan parasites infecting a wide range of animals, including humans. This study aimed to provide data on the occurrence, genetic characterization, and epidemiological significance of Sarcocystis spp. in cattle destined for human consumption in Romania. A total of 117 heart samples from slaughtered cattle in three southwestern Romanian counties (Dolj, Timiș, and Gorj) were analyzed in order to detect sarcocysts, using fresh examination microscopic techniques. Subsequently, the isolated sarcocysts and/or cyst fragments (5-15 per sample) from each infected animal were molecularly characterized. Overall, 17.9% (21/117) of the tested animals were found to be Sarcocystis spp. positive by microscopy. Genetic characterization of Sarcocystis spp. isolates, based on sequence analysis of the 18S rRNA gene, showed the presence of a single species, namely S. cruzi. No correlation was found (p > 0.05) between S. cruzi infection and the origin, age, breed, and gender of cattle, but the grazing farming system was positively associated (p=0.031) with the pathogen prevalence and can be considered a risk factor (OR = 3.6) in acquiring infection. To evaluate the possible public health risk, further investigation focused on the processing of other Sarcocystis-specific tissue matrices and evidence of human infections is recommended. This is the first study of bovine Sarcocystis infection in Romania.

Investigation of estrogen activity in the raw and treated waters of riverbank infiltration using a yeast estrogen screen and chemical analysis.

Exposure to various endocrine disrupting chemicals (EDCs) can lead to adverse effects on reproductive physiology and behavior in both animals and humans. An adequate strategy for the prevention of environmental contamination and eliminating the effects of them must be established. Chemicals with estrogenic activity were selected, and the effectiveness of their removal during the purification processes in two drinking water treatment plants (DWTPs) using riverbank infiltrated water was determined. Thirty-five water samples in two sampling campaigns throughout different seasons were collected and screened with a yeast estrogen test; furthermore, bisphenol A (BPA), 17ß-estradiol (E2) and ethinyl-estradiol (EE2) content were measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Our results confirm that estrogenic compounds are present in sewage effluents and raw surface river water of DWTPs. Very low estrogen activity and pg/L concentrations of BPA and E2 were detected during drinking water processing and occasionally in drinking water. Based on this study, applied riverbank filtration and water treatment procedures do not seem to be suitable for the total removal of estrogenic chemicals. Local contamination could play an important role in increasing the BPA content of the drinking water at the consumer endpoint.

Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016.

IntroductionThis paper reviews the current knowledge and understanding of Cryptosporidium spp. and Giardia spp. in humans, animals and the environment in 10 countries in the eastern part of Europe: Bosnia and Herzegovina, Croatia, Czech Republic, Estonia, Hungary, Latvia, Poland, Romania, Serbia and Slovenia. Methods: Published scientific papers and conference proceedings from the international and local literature, official national health service reports, national databases and doctoral theses in local languages were reviewed to provide an extensive overview on the epidemiology, diagnostics and research on these pathogens, as well as analyse knowledge gaps and areas for further research. Results:Cryptosporidium spp. and Giardia spp. were found to be common in eastern Europe, but the results from different countries are difficult to compare because of variations in reporting practices and detection methodologies used. Conclusion: Upgrading and making the diagnosis/detection procedures more uniform is recommended throughout the region. Public health authorities should actively work towards increasing reporting and standardising reporting practices as these prerequisites for the reported data to be valid and therefore necessary for appropriate control plans.

Survey of the Occurrence and Human Infective Potential of Giardia duodenalis and Cryptosporidium spp. in Wastewater and Different Surface Water Sources of Western Romania.

From the group of parasitic protozoa, Giardia and Cryptosporidium are the most common pathogens spread in surface water sources, representing a continuous threat to public health and water authorities. The aim of this survey was to assess the occurrence and human infective potential of these pathogens in treated wastewaters and different surface water sources. A total of 76 western Romanian water bodies in four counties (Arad, Bihor, Caraș-Severin and Timiș) were investigated, including the effluents of wastewater treatment plants (n = 11) and brooks (n = 19), irrigation channels (n = 8), lakes (n = 16), and ponds (n = 22). Water samples were collected through polyester microfiber filtration. Giardia cysts and Cryptosporidium oocysts were isolated using immunomagnetic separation, according to the US EPA 1623 method, followed by their identification and counting by immunofluorescence (IF) microscopy. All samples were screened through PCR-based techniques targeting the gdh gene for Giardia spp. and the 18S rRNA gene for Cryptosporidium spp., followed by sequencing of the positive results. Cryptosporidium-positive samples were subtyped based on sequence analysis of the GP60 gene. Giardia spp. was found in all tested water types with a cumulative detection rate of 90.1% in wastewaters, 26.3% in brooks, 37.5% in irrigation channels, 31.2% in lakes, and 36.4% in ponds. Except for ponds, all monitored water bodies harbored the Giardia duodenalis AII subassemblage with human infective potential. In addition, the ruminant origin assemblage E was widely distributed, and the domestic/wild canid-specific assemblage D was also recorded in a pond. Three (27.3%) wastewater samples were Cryptosporidium positive, and the identified species was the zoonotic Cryptosporidium parvum, with IIaA15G2R1 (n = 2) and IIdA18G1 subtypes. The results highlight that this threat to the public health must be brought to the attention of epidemiologists, health officials, and water authorities.

Giardia duodenalis and Cryptosporidium spp. as contaminant protozoa of the main rivers of western Romania: genetic characterization and public health potential of the isolates.

The objective of this study was to establish the prevalence, contamination level, and public health significance of Giardia duodenalis and Cryptosporidium spp. in the primary rivers of western Romania. A total of 53 sampling points in the 24 most important western Romanian rivers in four counties (Arad, Bihor, Caraș-Severin, and Timiș) were investigated from March to September 2016. Surface water samples were collected by microfiber filtration. Cryptosporidium and Giardia (oo)cysts were isolated using immunomagnetic separation (IMS) according to the USEPA 1623 method and, after staining with fluorescently labeled (FITC) monoclonal antibodies, were identified and counted under a microscope. The Cryptosporidium and Giardia (oo)cysts were identified to species and assemblage/sub-assemblage level through the nested PCR-RFLP procedure targeting the 18S ribosomal RNA and gdh genes, respectively. PCR-based techniques were utilized for all water samples. Overall, 22 samples (41.5%) were determined to be positive for Giardia cysts (ranging from 0.05 to 300 cysts per liter), and four samples (7.5%) tested positive for Cryptosporidium oocysts (0.17-48 oocysts/l). G. duodenalis was molecularly identified in 13 water samples (24.5%), indicating the presence of the sub-assemblage A-II (n = 12) and assemblage E (n = 1). PCR-RFLP showed that two samples (3.8%) contained Cryptosporidium DNA, and the identified species were Cryptosporidium parvum and Cryptosporidium canis. All positive results were successfully confirmed by DNA sequencing. Subtyping of the zoonotic C. parvum isolate based on sequence analysis of the GP60 gene revealed the occurrence of the IIaA16G1R1 subtype. The results of this study highlight considerable contamination of river waters with pathogenic Giardia spp. and Cryptosporidium spp., suggesting a potential risk for the public and animal health. This report presents the first extended published description of the presence of Giardia spp. and Cryptosporidium spp. in the aquatic environment in Romania.

Neglected waterborne parasitic protozoa and their detection in water.

Outbreak incidents raise the question of whether the less frequent aetiological agents of outbreaks are really less frequent in water. Alternatively, waterborne transmission could be relevant, but the lack of attention and rapid, sensitive methods to recover and detect the exogenous stages in water may keep them under-recognized. High quality information on the prevalence and detection of less frequent waterborne protozoa, such as Cyclospora cayetanensis, Toxoplasma gondii, Isospora belli, Balantidium coli, Blastocystis hominis, Entamoeba histolytica and other free-living amoebae (FLA), are not available. This present paper discusses the detection tools applied for the water surveillance of the neglected waterborne protozoa mentioned above and provides future perspectives.

Giardia and Cryptosporidium spp. dissemination during wastewater treatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loop mediated isothermal amplification (LAMP).

Environmental water samples from the Lower Rhine area in Germany were investigated via immunofluorescence assays (IFAs), nested polymerase chain reaction (nested PCR) and loop-mediated isothermal amplification (LAMP) to detect the presence of Giardia spp. (n=185) and Cryptosporidium spp. (n=227). The samples were concentrated through filtration or flocculation, and oocysts were purified via centrifugation through a sucrose density gradient. For all samples, IFA was performed first, followed by DNA extraction for the nested PCR and LAMP assays. Giardia cysts were detected in 105 samples (56.8%) by IFA, 62 samples (33.5%) by nested PCR and 79 samples (42.7%) by LAMP. Cryptosporidium spp. were detected in 69 samples (30.4%) by IFA, 95 samples (41.9%) by nested PCR and 99 samples (43.6%) by LAMP. According to these results, the three detection methods are complementary for monitoring Giardia and Cryptosporidium in environmental waters.

Detection and genotype analysis of Giardia duodenalis from asymptomatic Hungarian inhabitants and comparative findings in three distinct locations.

The transmission route of giardiasis not yet understood and why some infected individuals remain asymptomatic while others become quite ill. The drinking water quality is supposedly responsible for the prevalence of asymptomatic Giardia duodenalis infections in different areas, therefore asymptomatic giardiasis has been investigated in three water supply areas of Hungary: three hundred stool samples from inhabitants of Budapest, Füzér and Mátrafüred were examined by immunological and molecular methods for the presence of G. duodenalis infections. Individuals were asked to fill out a validated questionnaire at the time of stool collection and the interview covered demographic data, family life, education and travel history.In Budapest and in Mátrafüred in one stool sample G. duodenalis Assemblage A, whereas in Füzér once G. duodenalis Assemblage A, once Assemblage B and twice mixed infection were detected. We found higher prevalence rate of 4% of G. duodenalis infections of asymptomatic people in the village Füzér, where the removal of the Giardia cysts of the drinking water treatment plant was not effective. This study throws a light the need to look into the possibility of other risks of Giardia infections such as water transmission routes. To our knowledge, this is the first study evaluating the prevalence of G. duodenalis infections in asymptomatic persons in Hungary.

[Cryptosporidium and Giardia as water contaminant pathogens in Hungary]. / Cryptosporidium és Giardia mint vízszennyezo patogének Magyarországon.

INTRODUCTION: Many species of Cryptosporidium, and two assemlages of Giardia duodenalis cause typically acute diaorrhoea in human. The oocysts and cysts of these parasites excreted in faeces are capable of infecting other hosts and those are environmentally stable. AIM: The aims of the study were to evaluate the prevalence and genotypes of Cryptosporidium and Giardia species from different water sources as well as to monitor and characterize the (oo)cyst contamination sources in watersheds. In addition, an epidemiological study was performed in three selected settlements. METHOD: Wide range of modern epidemiological and molecular detection methods have been applied. RESULTS: (Oo)cysts densities were associated with water receiving effluents of sewage treatment plants or originating from a forest environment. It was confirmed, that cattle can be a source of Cryptosporidium oocysts at watersheds and aquatic birds can play a role in the environmental dissemination of these protozoa. The epidemiological study demonstrated a specific epidemiological situation, giving essential evidence about giardiasis in asymptomatic carriers. The applied novel detection technology was found to be cost effective and simple procedure for screening catchments to identify those that require further treatment and more detailed microscopic counts. CONCLUSIONS: The presented results contribute to a better understanding the epidemiology and relevance of waterborne parasites, their surveillance and performance of future control measures to prevent waterborne infections in Hungary.

Giardia taxonomy, phylogeny and epidemiology: Facts and open questions.

Giardia duodenalis (synonymous Giardia lamblia and Giardia intestinalis) is a flagellated protozoan parasite that reproduces in the small intestine causing giardiasis. It is a cosmopolitan pathogen with a very wide host range, including domestic and wild animal species, as well as human beings. In this paper the current knowledge about the taxonomy and phylogeny of G. duodenalis is summarized from the international literature and data on the detection and epidemiology are also reviewed concentrating on the last 20 years. Authors highlighted the current knowledge and some aspects on G. duodenalis in particular, water transmission and in vitro cultivation. The review sheds light on the difficulties of the strain differentiation and multilocus molecular analysis of Giardia strains especially when applied to water samples containing low numbers of cysts and components complicating the problem of tracking sources of contamination. Genetic elements determining or conferring traits such as infectivity, pathogenicity, virulence, and immune interaction contributing to clearance are currently not well established, if at all. These should be useful and important topics for future research.

First description of Cryptosporidium bovis in Japan and diagnosis and genotyping of Cryptosporidium spp. in diarrheic pre-weaned calves in Hokkaido.

Eighty fecal samples from pre-weaned calves with diarrhea were collected in the Tokachi area in Northern Japan to investigate the prevalence of Cryptosporidium species in such animals. Oocysts from fecal samples collected from each animal were concentrated using sucrose gradient centrifugation. Genomic DNA was extracted from each sample and processed by nested PCR to amplify the partial SSU rRNA gene of Cryptosporidium. Cryptosporidium infections were detected in 75% of the samples. Sequence analysis was performed on all positive samples. Phylogenetic analysis of 33 successfully sequenced isolates of the SSUrRNA PCR products revealed all but one were Cryptosporidium parvum infections. The remaining single case was Cryptosporidium bovis. These findings suggest that C. parvum is prevalent in diarrheic pre-weaned calves and can be a source of cryptosporidial infections for humans and animals in Hokkaido.

Genetic polymorphism in Cryptosporidium species: an update.

Cryptosporidia, widely distributed protozoan parasites of vertebrates, have attracted increasing interest due to several serious waterborne outbreaks, the life-threatening nature of infection in immunocompromised patients, and the realization of economic losses caused by these pathogens in livestock. Genetic polymorphism within Cryptosporidium species is being detected at a continuously growing rate, owing to the widespread use of modern molecular techniques. The aim of this paper is to review the current status of taxonomy, genotyping and molecular phylogeny of Cryptosporidium species. To this date, 20 Cryptosporidium species have been recognized. Two named species of Cryptosporidium have been found in fish, 1 in amphibians, 2 in reptiles, 3 in birds, and 12 in mammals. Nearly 61 Cryptosporidium genotypes with uncertain species status have been found based on SSUrRNA sequences. The gp-60 gene showed a high degree of sequence polymorphism among isolates of Cryptosporidium species and several subtype groups and subgenotypes have been identified, of which the Cryptosporidium parvum IIa and IId subtype groups were found to be zoonotic. This review describes considerable progress in the identification, genetic characterization, and strain differentiation of Cryptosporidium over the last 20 years. All the valid species, genotypes and zoonotic subtypes of Cryptosporidium reported in the international literature are included in this paper with respect to the taxonomy, epidemiology, transmission and morphologic-genetic information for each species.

The role of aquatic birds in the environmental dissemination of human pathogenic Giardia duodenalis cysts and Cryptosporidium oocysts in Hungary.

Fecal samples were taken from 132 (103 wild and 29 domestic) aquatic birds on selected areas in Hungary from February 2008 to March 2008. Cryptosporidium oocysts and Giardia cysts were purified from the samples and were viewed via fluorescent antibody staining. Molecular detection tools, such as PCR-sequencing and Loop mediated isothermal amplification (LAMP) were used in order to determine the Cryptosporidium species and Giardia duodenalis assemblages present. All together 6 (5.8%) and 6 (5.8%) samples out of the 103 wild bird samples and 4 (13%) and 7 (24%) samples out of the 29 domestic bird samples have been found to be Cryptosporidium and G. duodenalis positive respectively. The results of this study indicate that aquatic ducks, geese, coot and cormorant can play role in the environmental dissemination of human pathogenic Giardia cysts and Cryptosporidium oocysts in Hungary. To our knowledge, this is the first description of Cryptosporidium sp. in Anser fabalis and Anser anser, furthermore Giardia sp. in Fulica atra, A. fabalis and P. carbo and the first PCR-sequence confirmed detection of C. parvum in A. platyrhynchos and F. atra, G. duodenalis Assemblage A in A. strepera and G. duodenalis Assemblage B in A. anser.

Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes.

We investigated the prevalence of Giardia and Cryptosporidium species and analysed the genotypes in 36 samples collected from different water sources and various geographic areas in Hungary. Samples were collected from drinking water and sewage treatment plants and from the recreation area of Lake Balaton. The (oo)cysts were purified according to the US EPA 1623 method and they were detected by immunofluorescence test (IFT). Genomic DNA was extracted from all samples and then the GDH target gene for Giardia and the SSUrDNA for both Giardia and for Cryptosporidium species were amplified by PCR. 24 out of 36 samples (67%) were Giardia positive and 15 (42%) were Cryptosporidium positive by IFT. PCR confirmed that 13 out of 36 samples (36%) were Giardia positive and 10 (28%) contained Cryptosporidium. Twelve Giardia and two Cryptosporidium PCR products were successfully sequenced. In seven samples G. lamblia Assemblage A and in one sample Assemblage B and in four cases Assemblages A and B have been found. In one sample C. parvum and in the other separate sample C. meleagridis were detected. Sequence analysis revealed a new subtype of G. duodenalis complex, clustered close to the Assemblage A group. This study provides the first report on simultaneous detection and genotyping of G. duodenalis and Cryptosporidium species from water supplies in Hungary.

Prevalence and genotyping of Cryptosporidium species from farm animals in Mongolia.

The presence of Cryptosporidium oocysts in 460 animals (439 cattle, 16 kids, and 5 sheep) of Tuv-aimak Mongolian district was investigated by IFT. Cryptosporidium oocysts were found in 116 (26.4%) cattle. Out of the 116 IFT positive samples, 47 were further purified by IMS, investigated by PCR and 11 were found positive. The species and/or genotypes were determined by nested PCR-RFLP and sequence analysis of a fragment of the SSU rRNA gene. The results indicated the presence of Cryptosporidium andersoni in the sequenced samples and C. bovis in two samples as a common infection. No Cryptosporidium oocysts were found in fecal specimens collected from sheep and goats. The present work reports the first data on Cryptosporidium species in animals from Mongolia. Further studies are necessary to understand the epidemiology and transmission of Cryptosporidium in domestic animals in Mongolia.

Molecular characterization of Cryptosporidium from animal sources in Qinghai province of China.

The presence of Cryptosporidium oocysts in 20 zoo animals of the Xining Zoo, 16 farm yaks and 42 farm goats in Qinghai province, China was investigated by an immunofluorescence test (IFT). The species and/or genotypes were determined by nested polymerase chain reaction (PCR) and sequence analysis of a fragment of the small subunit (SSU) rRNA gene. Cryptosporidium oocysts were found in 16 zoo animals, 2 yaks, and 15 goats by IFT. The IFT positive samples were further investigated by PCR, and 16 of them were found to be positive by that method also. Sequence analysis of the PCR products derived from Cryptosporidium oocysts from Black leopard (Panthera pardus), Heijing He (Grus nigricollis), Barbary sheep (Ammotragus lervia), Takin (Budorcas taxicolor), Lesser panda (Ailurus fulgens), and White-eared pheasant (Crossoptilon crossoptilon) fecal samples matched that of Cryptosporidium parvum mouse genotype. Sequence analyses of other PCR products were consistent with cervine genotype Cryptosporidium from Ibex (Capra ibex), a novel Cryptosporidium genotype from a wild yak (Bos mutus), C. bovis-like genotype from one goat sample and also a novel Cryptosporidium genotype from one other separate goat sample. The present work reports the first data on Cryptosporidium infections in animals from the Qinghai province of mountainous central western China and the first findings of the 'cervine' genotype in Capra ibex, C. bovis-like genotype and the new Cryptosporidium spp. in farm goat and in wild yak.