Survey and first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea, Turkey.

Infection with Acanthamoeba spp. may result in granulomatous amoebic encephalitis and Acanthamoeba keratitis. Water is an important habitat where Acanthamoeba species thrive. Therefore, studying the occurrence of this free-living amoeba in water sources will help understand the infection dynamics. The aim of the study was to survey and report on the presence of Acanthamoeba spp. in water resources from the Ordu and Giresun provinces in Black Sea. Acanthamoeba spp. was found in 1/17 natural spring water samples from Ordu and in 2/18 from Giresun. Acanthamoeba species were not detected in any of the investigated tap water samples. Sequencing of the (SSU) rDNA gene resulted in the identification of haplotype I (Acanthamoeba genotype: KJ094684). T4 (8.6%) was the only isolated genotype in both Ordu and Giresun provinces. This is the first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea. The occurrence of Acanthamoeba species in natural spring water sources should be considered as a potential risk for human infection, especially to high-risk populations.

Acanthamoeba spp. in river water samples from the Black Sea region, Turkey.

The present study aims to investigate the occurrence of free living amoeba (FLA) in water resources (rivers and tap water) in Samsun in the Black Sea. The presence of Acanthamoeba spp. was confirmed in 98 of 192 water samples collected from 32 sites of Samsun province (Samsun centre, Terme, Carsamba, Tekkekoy, Bafra) by PCR. Acanthamoeba spp. were found in 15/36 river samples from Samsun, in 58/90 from Terme, in 12/30 from Carsamba, in 7/18 from Tekkekoy and in 6/18 from Bafra. No Acanthamoeba species were detected in tap water samples. The highest rate in river waters contaminated with Acanthamoeba species was in Terme followed by Samsun centre (41.7%), Carsamba (40%), Tekkekoy (38.9%) and Bafra districts (33.3%), respectively. The result of the subsequent sequence analysis showed Haplotype I (A. triangularis) in 5%, Haplotype II (A. polyphaga) in 29.6%, Haplotype III (Acanthamoeba spp.) in 62% and Haplotype IV (A. lenticulata) in 3%. The most common genotype was Acanthamoeba T4 (Acanthamoeba spp., A. polyphaga, A. triangularis) and T5 genotype was also found in 3%. The T4 genotype is the most common genotype associated with Acanthamoeba keratitis (AK) worldwide; therefore, humans and animals living in the area are at risk after contact with such waters.

Investigation of Blastocystis Subspecies in Water Samples Collected from Ordu Province

Objective: The aim of the study was to determine the prevalence of Blastocystis subspecies in water samples collected from Ordu province. Methods: Seventy-five surface water samples and 25 drinking water samples were collected from Ordu and its boroughs. The samples were flocculated by aluminum sulphate and concentrated by sucrose gradient method. Small subunit ribosomal RNA (SSU rRNA) gene was amplified by polymerase chain reaction (PCR). Sequence analysis was performed on the positive PCR products and the base sequences were aligned using Bioedit. Phylogeny trees were drawn and Blastocystis subspecies were identified. Results: Four out of the 100 water samples were positive for Blastocystis spp. No positivity was found in the drinking water. Blastocystis ST-1 subspecies was detected in Bülbül River, Kacali River and Bolaman Stream. Blastocystis ST-3 subspecies were found in Karabalçik River. The lowest genetic distance was found between ST-1 subspecies and the samples from Bülbül River, Kacali River and Bolaman Stream. The nucleotide similarities between them were 98.8%, 76.6 and 98.8%, respectively. The lowest genetic distance was found between Karabalçik River and ST-3 subspecies and the nucleotide similarity between them was 99.1%. Conclusion: This is the first study on the presence of Blastocystis spp. in the surface water and drinking water samples in Ordu province of the Black Sea area in Turkey.

Amebicidal effects of fenugreek (Trigonella foenum-graecum) against Acanthamoeba cysts.

Trigonella foenum-graecum L. (TF) is known to the public as a chest emollient, mucous expectorant, laxative and is used to prevent maturation of boils and diabetes since ancient times. In this study, we aimed to determine the amebicidal effects against Acanthamoeba cysts. Plant extracts were prepared at concentrations of 1, 2, 4, 8, 16, and 32 mg/ml and were placed in a hemocytometer with cell counts 22 × 106 cell/ml. The fatty acid profiles of TF seeds were determined. Standard Acanthamoeba cysts were added and incubated at 25°C. The viability of the parasite was checked and recorded at hours 3, 24, 48, 72, 96, and 102. The values of lethal concentration doses (LD50 and LD90) were calculated using probit analysis. This study revealed that T. foenum-graecum prevented proliferation of the parasite at certain times. However, further for in vivo and controlled experimental studies are needed in order to find out how to use this plant as medication.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

Molecular identification of Blastocystis sp. subtypes in water samples collected from Black sea, Turkey.

The aim of this study was to identify the subtypes of Blastocystis sp. and complete a phylogenetic analysis of 268 water samples that were collected from the Samsun, Amasya and Sinop Provinces of the Black Sea in Turkey, between the years 2011 and 2014. Blastocystis sp. was investigated in 48 uncultured sea water samples that were collected from 4 sites within the Sinop Province. A total of 100 river water samples were collected from 37 sites in the Samsun Province and 120 river water samples were collected from 10 sampling sites within the Amasya Province. The small subunit (SSU) rDNA gene Polymerase chain reaction (PCR) were performed for the detection of Blastocytis sp. and the PCR-positive samples were sequenced. Subsequently, the (SSU) rDNA sequences were aligned by Bioedit and phylogenetic trees were constructed for Blastocystis with reference to the genotypes from GenBank. Blastocystis sp. were found in 3 out of the 75 (4%) river water samples that were collected from the Samsun Province. Six of the 120 (5%) river water samples and 1 out of the 48 (2%) seawater samples were positive for Blastocystis in the Amasya and Sinop Provinces. There were two different subtypes (ST; 1 and 3) found from sequencing all of the samples from the investigated sites. Two and one PCR products were found to be positive for ST1 and ST3 from the different samples collected within the Samsun Province. Two and 4 PCR products from the Amasya Province were ST1 and ST3, respectively and only one sample from the Sinop Province was found to be positive for ST1. This is the first report to identify and report the occurrence of Blastocystis subtypes within the Black Sea of Turkey.

Presence of Parasites in Environmental Waters in Samsun and Its Districts.

OBJECTIVE: The aim of this study was to detect the presence of parasites in environmental waters in Samsun and its districts. METHODS: At the center of Samsun, 13 stations were determined. The research was performed between March 2012 and February 2013, and every month, water samples were collected on the dates stated. The samples were stained with Kinyoun acid-fast, modified trichrome, and trichrome dyes after examining with the direct bond. The preparations were evaluated in terms of parasitologic under a light microscope. RESULTS: Totally, 180 of 228 water samples analyzed were from streams; of these, 48 were drinking water samples. The following were found: 142 Giardia spp., 132 Cryptosporidium spp., 56 Cyclospora spp., 38 microsporidia, 47 Blastocystis spp., 38 Entamoeba coli cysts, 18 Dientamoeba, 9 Chilomastix, 9 Strongyloides spp., and 6 hookworms. CONCLUSION: The widespread use of animal husbandry and agriculture in the region and the use of stream surroundings as a grazing area increase the presence of some determined protozoa during a certain period. Parasitological studies in humans and animals in the region should be conducted, and control programs should be applied.

Genotyping of Cryptosporidium spp. in environmental water in Turkey.

This research was undertaken to study the molecular detection and characterization of Cryptosporidium spp. in environmental water sources at Samsun and Giresun Provinces of The Black Sea in Turkey. Two-hundred forty and one-hundred eighty environmental samples were collected from a total of twenty and twenty-five sampling sites of Giresun and Samsun Provinces. One hundred twenty untreated drinking water samples were also detected for Cryptosporidium spp. in both investigated areas. 101 (%42), 92 (%38.3) of 240 and 74 (41.1%), 70 (38.8%) of 180 environmental samples have been found positive for Cryptosporidium spp. by Loop mediated isothermal amplification (LAMP) targeting the S-adenosyl-L-methionine synthetase (SAM) gene and nested PCR targeting small subunit (SSU)rRNA gene in Samsun and Giresun Provinces, respectively. Of the tested untreated drinking water samples collected from the investigated area, one sample was positive for Cryptosporidium spp. Six and twelve samples were clearly sequenced for the Cryptosporidium (SSU)rRNA gene among the highest positive samples selected from each of the twenty and twenty-five sampling sites of Giresun and Samsun Provinces, respectively. Genetic characterization of Cryptosporidium isolates from water samples represented Cryptosporidium bovis for five samples, Cryptosporidium parvum for six samples and one sample for Cryptosporidium felis in Samsun Province, where C. parvum for five samples and C. bovis for one sample were sequenced in Giresun Province. According to accessible information sources, this is the first research about genotyping of Cryptosporidium spp. in water samples collected from Samsun and Giresun Provinces of Turkey.

Occurency of Giardia duodenalis assemblages in river water sources of Black Sea, Turkey.

A total of 420 environmental water samples and 120 drinking water samples from 45 different sampling sites of the Black Sea in Turkey were collected between 2012 and 2014. Genomic DNA was isolated from all the investigated water samples and comparativelly analyzed by Loop-mediated isothermal amplification (LAMP) of the elongation factor 1 Alfa (EF1α) gene, and by nested Polymerase Chain Reaction (nPCR) of the small subunit (SSU) rRNA and semi-nested PCR (snPCR) of the glutamate dehydrogenase gene (GDH). 141 (58.7%), 125 (52.1%) and 120 (50%) samples respectivelly were positive by each method. Out of 240 environmental samples collected from 25 sites of Samsun Province have been found positive for G. duodenalis by LAMP, nPCR and snPCR, respectively. 55 (30.5%), 50 (27.8%) and 47 (26.1%) of 180 environmental samples collected from 20 other sampling sites of Giresun Province were positive for Giardia by LAMP, nPCR and snPCR, respectively. Five PCR products from different samples of the Giresun Province and 10 other samples from the Samsun Province were found positive for G. duodenalis assemblage B. Five PCR products from Giresun Province and 5 samples from Samsun Province were found positive for G. duodenalis assemblage A. This is the first report about G. duodenalis assemblages A and B from water samples investigations in Black Sea of Turkey.

PREVALENCE OF DEMODEX ECTOPARASITES AMONG HUMANS IN ORDU PROVINCE IN TURKEY.

Demodex folliculorum and Demodex brevis are common ectoparasites in humans. Demodex parasite infestations have not been determined in the province of Ordu. We determined the prevalence of Demodex species among humans in Ordu Provience, Turkey. Seven hundred ninety-nine subjects (438 males and 361 females) aged ≥ 18 years living in the central districts of Ordu Province, Turkey, were selected using the World Health Organization cluster sampling method. A superficial skin biopsy of the face was obtained from each subject. Six hundred sixty-nine subjects (83.7%) had a Demodex parasite. Factors significantly associated with the presence of Demodex infestation were: female gender, employment in the private sector, people who only occasionally wash their face and district of residence. Since Demodex ectoparasites were common in Ordu Province, it is suggested that the diagnosis and treatment of this ectoparasite should be carried out in the hospitals of this region.

[Investigation on Toxoplasma gondii by polymerase chain reaction and loop-mediated isothermal amplification in water samples from Giresun, Turkey]. / Giresun'da su örneklerinde Toxoplasma gondii'nin polimeraz zincir reaksiyonu ve ilmige dayali izotermal amplifikasyon yöntemleriyle arastirilmasi

Toxoplasmosis is generally asymptomatic in immunocompetent subjects, however serious manifestations of the disease may develop in immunocompromised patients and in pregnant women. The mean Toxoplasma gondii seroprevalence which is approximately 40% in Turkish population, indicates the high risk for the development of acute toxoplasmosis in those cases. One of the transmission ways of T.gondii is the consumption of contaminated water. The aim of this study was to detect the presence of T.gondii in the environmental and drinking water samples by using standard polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods. A total of 96 water samples, of them 76 were environmental water samples collected from creeks in Giresun province center and its various districts (Piraziz, Bulancak, Kesap, Espiye) and 20 from drinking water samples, were included in the study. The samples were precipitated by the aluminium sulfate method and DNAs were isolated from the pellets formed with the sucrose gradient method. PCR and LAMP were applied to the isolated DNAs, by the use of primers F3 and B3 specific to B1 gene of the parasite. In our study, T.gondii was detected in none of the drinking water samples by PCR and LAMP, however T.gondii DNAs were positive in 13.2% (10/76) of the environmental water samples. Both of the methods yielded the same results in those samples. The stations that were positive for T.gondii were Aksu creek in Giresun province center; Gelivera creek in Espiye; Yolagzi, Kesap, Kesap Login Bridge creeks in Kesap; Bulancak, Karadere and Incivez creeks in Bulancak; Piraziz and Çayiragzi creeks in Piraziz counties. Resistance of T.gondii to chlorination and the inadequacy of the filtration processes, create a serious threat among people in contact with rivers, sea and drinking waters particularly in areas with high humidity. As far as the national literature was considered, no report about the water-borne T.gondii infections were detected in Giresun province of Black Sea region, Turkey. Thus this study will aid to the literature related to water-borne T.gondii infections. The results of this study emphasizes the essential need for appropriate disinfection procedures and purification processes before the discharge of waste water to prevent the cases of water-borne toxoplasmosis.

[The epidemiology of demodex mites at the college students living in dormitories in the city of Ordu]. / Ordu Ilinde Yurtlarda Kalan Üniversite Ögrencilerinde Demodex Türlerinin Epidomiyolojisi.

OBJECTIVE: Demodex folliculorum and Demodex brevis can cause different skin and eye symptoms. There are indications showing that the prevalence of these parasites is higher in public places. In this direction, the study was aimed to determine the prevalence of demodex in university students, inhabiting in dormitories. METHODS: The study consisted of 300 men and women, college students who were staying in the dormitories in the city of Ordu. Random sampling method was used in the sample selection. Each participating student had to sign a patient's consent form, before samples were taken using standard superficial skin biopsies from the faces of the patients. The samples were embedded in Entellan mounting solution and examined under the light microscope. RESULTS: Samples were taken from 300 college students (170 males and 130 females) aged between 18-30 years, and in 110 (37%) of them, demodex mites were found. No significant differences were found between gender, age, type of skin or skin care, and Demodex incidence. CONCLUSION: Demodex mites are very prevalent in college students studying in Ordu.

Detection of Cryptosporidium species in the sea and tap water samples of Black Sea, Turkey.

The aim of this study was to evaluate Cryptosporidium spp. contamination of sea and tap water samples from Sinop and Ordu Provinces, Black Sea, Turkey. The samples (10 L) were collected in spring, summer, autumn, and winter in 2011. A total of 128 water samples was analyzed using an immunofluorescence test (IFT), as well as loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Cryptosporidium spp. oocysts were detected by IFT in 43 of the 70 samples (61.4%; 1-40 oocysts per 0.5 L) and 35 of the 58 samples (60.3%; 1-23 oocysts per 0.5 L) in the sea water samples from Ordu and Sinop, respectively. The highest number of oocysts by IFT were detected in spring and winter in Ordu and Sinop, respectively. The results of the S-adenosylmethionine synthetase (SAM) gene LAMP assays were 65.5% positive for Cryptosporidium parvum , Cryptosporidium hominis , and Cryptosporidium meleagridis in all examined samples, while the SSUrRNA gene nested PCR assay was 31.0% positive. Six C. parvum nested PCR products from all positive samples were successfully sequenced.

[Diagnosis of protozoa by loop-mediated isothermal amplification: (LAMP)]. / Protozoonlarin tanisinda ilmige Dayali Izotermal Çogaltma Yöntemi (LAMP).

Plasmodium, Isospora, Toxoplasma, Babesia and Cryptosporidium are parasites which have a significant role in human health. The location in which the protozoon settles in the body, the way that it leaves the host, and the sample which is send from the clinic are the elements of the general methods used in the diagnosis of the ailments caused from protozoan. Classical methods are not adequate for the identification of some protozoon; also, molecular methods have to be used in designating the distinctions between the species. In recent years, one of the molecular methods which has been used frequently in the identification of protozoon is the technique of LAMP. With the aid of the LAMP technique, from which it is possible to obtain reliable outcomes without the contribution of technical skills and professional equipment, in constant temperature it is possible to have a great number of copies from the targeted DNA in a short period. The aim of this collation is to give information about the usage of the technique of LAMP in the identification of the protozoa which are important for human health and the comparison between the technique of LAMP and other molecular methods.

Transient expression of red and yellow fluorescent protein vectors in HCT-8 cells infected with Cryptosporidium parvum.

Electroporation in combination with the red and yellow fluorescent protein (RFP and YFP, respectively) reporter systems results in transient gene expression in HCT-8 cell lines infected and uninfected with Cryptosporidium parvum. Conditions are described allowing efficient electroporation of HCT-8 cell monolayers in a commercial electroporation device (180 kV/cm voltage, 250 microF capacitance, and 0 ohm (ohm) resistance resulting in a time constant of 0.4 ms.). Fluorescent microscopy showed that uninfected HCT-8 cell monolayers achieved higher gene transfer and expression efficiencies than HCT-8 cells infected with C. parvum under similar conditions. Real-time melting curve and quantitation analysis of reverse transcription polymerase chain reaction amplicons are presented for the detection of all mRNA sample levels. Our findings demonstrate that C. parvum infection of HCT-8 cells may negatively affect the transient expression of RFP and YFP plasmids. The focus of this study was to achieve transient expression of reporter genes in HCT-8 cell lines and provide the basis for further analyses of gene regulation in protozoan. These approaches may provide to understanding the feasibility of gene transfer and expression efficiencies HCT-8 cell cultures by RFP and YFP containing the CMV promoter; they could serve as tools for gene transfer in mammalian cell.