Determination of the anti-protozoal activity of medicinal agents using the phenomenon of plaque formation by Acanthamoeba spp.

Representatives of the genus Acanthamoeba are among the most widespread protists in the environment. They have a ubiquitous distribution and can sometimes cause quite serious pathologies in humans. The treatment ofp rotozoal infections caused by free-living amoebae is currently limited and often unsuccessful. In the presented investigation, amebicidal activity was determined against both the trophozoites and cysts of Acanthamoeba spp., which were isolated during the microbiological examination of environmental objects. The inhibitory activity of drugs in vitro was determined using the authors' proposed method, which is based on the plaque formation phenomenon: this is initiated by free-living amoebae when cultured in agar containing the bacteria Cellulosimicrobium sp. strain bent-1. Based on a series of experimental studies, the paper proposes a reliable and inexpensive method for determining the anti-protozoal activity of medicinal agents, which will significantly complement the current screening method system when studying existing drugs, or new drugs during their development stage.

Cutaneous larva migrans: A One Health Perspective on Familial Infection Among Tourists Returning from Southeast Asia.

Cutaneous larva migrans (CLM) is a dermatosis caused by accidental infestation with animal hookworms and is widely distributed in tropical and subtropical regions. Humans become infected when their skin comes into contact with soil contaminated with dog faeces. The filariform larvae penetrate and burrow into human skin, causing a condition known as "creeping eruption". We describe a case, well-documented by photos, of CLM infection in a family of three who returned from Thailand.

Asymptomatic carrier of Babesia spp. among blood donors - epidemiological situation in Poland.

Introduction: Babesiosis is caused by one of several Babesia species. In Europe, B. divergens predominates in humans, while in North America it is B. microti. Babesia spp. infection in donors with a disease-free course of infection can be a major problem in blood recipients. A recipient with impaired immune system functions is at risk of full-blown development of the disease. In Poland and in most countries of the world, blood donors are not routinely tested for Babesia spp. infection. In our previous study, we detected Babesia venatorum DNA in blood donors, which was the reason for expanding the study to include more test subjects. Objective: The aim of this study was an attempt at estimating the prevalence of asymptomatic infection with Babesia spp. among blood donors from the Regional Centres for Blood Donation and Blood Treatment in Warsaw and Wroclaw. Materials and methods: The material for the study was whole blood from regular blood donors from two Regional Centre for Blood Donation and Blood Treatment in Warsaw and Wroclaw. Whole blood samples from 1,067 blood donors collected in June-July 2022 were analyzed. Blood collected directly from the donor during the blood donation procedure. All persons qualified by a doctor as a donor were selected for the study, regardless of age and sex. All subjects were informed in detail about the purpose of the study and gave their written consent. Isolation was made by using the Chelex 100 chelating resin, followed by the studying of the genetic material using the qPCR reaction. The results were analysed based on the amplification curve. Results: The protozoan Babesia spp. was not detected in the blood samples. Conclusions: The risk of blood-borne babesiosis is extremely low in Poland.

Helminth Community Structure of Tits Cyanistes caeruleus and Parus major (Paridae) during Their Autumn Migration on the Southern Baltic Coast.

The research problem undertaken in this study is to determine the scale of infection of Eurasian blue tit Cyanistes caeruleus and Great tit Parus major and the biological diversity of their internal parasites, helminths. The aim of the study is to gain new knowledge about the structure of the helminth communities of the Eurasian blue tit and Great tit on the southern coast of the Baltic Sea during autumn migration to their wintering grounds. Helminths of tits were collected in 2008-2012 on the southern coast of the Baltic Sea in Poland. PAST v. 2.11 software was used for the calculations. Barcoding DNA was used to identify trematodes initially classified based on morphological characters to the genera Leucochloridium and Urogonimus. Cestodes Anonchotaenia globata were recorded for the first time in Poland. The Eurasian blue tit is a new host in Poland for three species of helminths: cestode Monosertum parinum and filarial nematodes, Cardiofilaria pavlovskyi, and Diplotriaena henryi. The Great tit is a new host in Poland for trematode Urogonimus macrostomus, cestode A. globata and M. parinum, and filarial nematode Diplotriaena obtusa. The nematode C. pavlovskyi was the species most frequently recorded in both host species. A high degree of similarity was found between the component communities and infracommunities of helminths in Eurasian blue tit and Great tit. The new information provided in this study has increased our knowledge of the transmission of helminths in Central Europe.

Improved Blastocystis spp. detection method using swabs with Amies transport medium and charcoal.

Blastocystis is one of the most frequently detected protozoa in the human large intestine. One of the most effective and cheap methods for detecting Blastocystis in faeces is culture on a special medium in anaerobic conditions. Sampling faeces using traditional containers and their transport to the laboratory has certain limitations: a sample taken in this way should reach the laboratory relatively quickly, moreover, some patients are uncomfortable during sampling and protection of material in this way. We propose utilizing a swab for sampling and transportation of the faeces samples to be examined for Blastocystis instead of using traditional containers. We believe this is an excellent method allowing the material to be transported over longer distances without additional, and sometimes expensive, safety measures, and at the same time permitting the possibility of obtaining living cells after a relatively long period of storage.

Astroblastocystis nom. nov. - a new replacement name for Blastocystis Jaekel, 1918 (Echinodermata, Parablastoidea).

The generic name Blastocystis has been proposed at least twice for different organisms for which nomenclature is governed by the International Code of Zoological Nomenclature. Blastocystis Jaekel, 1918, is a junior homonym of the generic name Blastocystis Alexeieff, 1911. We propose the following Astroblastocystis nom. nov. as a new replacement name (neonym) for Blastocystis Jaekel, 1918.

Molecular Study of Echinococcus granulosus Cestodes in Ukraine and the First Genetic Identification of Echinococcus granulosus Sensu Stricto (G1 Genotype) in the Country.

INTRODUCTION: Cystic echinococcosis is a globally distributed zoonotic disease of great medical and veterinary importance, which is caused by the cestode Echinococcus granulosus sensu lato. In Ukraine, two areas of the prominent circulation of the parasite are established, the southern steppe zone with sheep as the main transmitter, and the northern forest-steppe zone and Polissia, where pigs are mainly responsible for maintaining the E. granulosus transmission. METHODS: Given that only a few studies have so far addressed the genetic diversity of the parasite in Ukraine, we have sequenced partial mitochondrial genes of cytochrome c oxidase 1 (789 bp), NADH dehydrogenase 1 (602 bp) and 12S rRNA (333-334 bp) in pig metacestodes from the Sumy region (farms close to Sumy, northeastern Ukraine) and the Kyiv region (a farm in Bila Tserkva, central Ukraine). RESULTS: Four isolates from four pigs in the Sumy region were identified as E. canadensis (G7 genotype), the major E. granulosus s.l. species circulating in Eastern Europe, including the three microvariants (G7A, G7B, G7C). Three isolates from the two pigs in the Kyiv region were classified as E. granulosus s.s. (G1 genotype), including one microvariant (G1A). CONCLUSION: To our knowledge, this is the first genetic record of E. granulosus s.s. with the presumed highest infectivity and virulence among the E. granulosus s.l. species in Ukraine. The finding has implications for public health as local control programmes should take into consideration different development rate of this parasite in dogs and the greater risk of the species for human infection.

Intestinal Helminth Communities of Grey Partridge Perdix perdix and Common Pheasant Phasianus colchicus in Poland.

The aim of this study was to describe the morphology and means of identification of helminths in native partridges (65) and introduced pheasants (32) in Poland and to determine the level of intestinal infection of these birds by helminths using parasitological and ecological indices. The birds were acquired during the hunting season in the years 2015-2017. Nematodes, Capillaria phasianina, cestodes, Railietina friedbergeri, and one trematode, Brachylaima sp. were recorded for the first time in partridges in Poland. Our findings indicate that parasites are more prevalent in pheasants (prevalence 70.4%) than in partridges (prevalence 50.0%). The component community and infracommunity of parasites of partridges are more diverse (Simpson's diversity index: 0.63 and mean Brillouin diversity index: 0.10 ± 0.17) and less dominated by a single parasite species (Capillaria sp., Berger-Parker dominance index: 0.53) than the pheasant parasite community (Simpson's diversity index: 0.07, mean Brillouin diversity index: 0.005 ± 0.02, dominant species Heterakis gallinarum, Berger-Parker dominance index: 0.96). There were statistically significant differences between partridges and pheasants in the Brillouin diversity index and in the prevalence of Heterakis gallinarum (55.6% in pheasants vs. 19.0 in partridges). There were significant differences between wild and farmed partridges in the prevalence of infection by Capillaria sp. (4.3% vs. 37.5%) and H. gallinarum (39.1 vs. 6.2%). In conclusion, the pheasant was shown to be a reservoir, carrier, and shedder of nematodes, which may increase the risk of infection in partridges.

Occurrence of Echinococcus spp. in red foxes and wolves in the protected area of the Tatra National Park in southern Poland - a threat to human health.

INTRODUCTION: Echinococcus multilocularis has been endemic in red foxes in eastern and central parts of Europe, and E. granulosus s. l. identified in wolves in some countries. In recent years, wolves hale emerged as potentially important definitive hosts of E. multilocularis. OBJECTIVE: This aim of the survey was to record indirectly using nested-PCR test with faecal samples the presence of Echinococcus multilocularis and E. granulosus s. l. in the two species of wild canids in the protected area of the Tatra National Park (TNP) in Western Carpathian, southern mountainous part of Poland. MATERIAL AND METHODS: From February to June 2019, experienced staff of TNP randomly collected fox and wolf faeces on and off hiking trails at altitudes from 850 m to 2,000 m above sea level. In total, 91 faecal samples from red foxes and 19 from wolves were collected. Genomic DNA was obtained by direct extraction from faecal samples using a commercial kit, and from taeniid eggs retrieved from the same samples after flotation. RESULTS: A nested PCR screening of 91 red fox faeces indicated the prevalence of E. multilocularis of 4.4%. Positive samples were confirmed by sequencing parts of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Neither genomic DNA of E. multilocularis nor of E. granulosus s.l. was obtained from 19 wolves faeces, nor from taeniid eggs retrieved from these samples by initial flotation. CONCLUSIONS: The current results show that humans might be exposed to a risk of fox tapeworm infection in nature, even at high altitude inan alpine zone, in an environment contaminated by roaming red foxes encouraged by food leftovers on mountain trails.

Blastocystis occurrence and subtype diversity in wild European terrestrial mammals - The case of Bialowieza Primeval Forest (NE Poland).

Blastocystis cf. Blastocystis hominis is the most common unicellular parasite found in human and animal intestines. Little is known about the life cycle, transmission and mechanisms of pathogenesis for this parasite. The aim of this study was to obtain new data on the genetic diversity of Blastocystis in selected species of mammals found in the Bialowieza Primeval Forest (BPF), Poland, the best preserved lowland forest in temperate Europe. 113 faecal samples were collected in the period 2018-2020 from seven species of wild mammals occurring within the Polish part of BPF and its surroundings. Blastocystis was detected by molecular amplification and sequencing the small subunit rRNA gene. The overall prevalence of Blastocystis in animals was 8.9%. A larger number of stool samples (90) were collected from European bison and only for this species was it possible to calculate the prevalence of infection (5.6%). The isolates obtained from European bison were classified as ST1, ST3, ST5, ST7. Blastocystis was also detected in the grey wolf (Canis lupus) (ST5), wild boar (Sus scrofa) (ST5) and red deer (Cervus elaphus) (ST1). In conclusion, for the first time we have demonstrated the presence of Blastocystis in wild European bison. Preliminary studies have shown that Blastocystis is present in at least 4 species of wild mammals in the BPF area and that it exhibits great genetic diversity.

The first case of periorbital human dirofilariasis in the Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.

Sparganosis (Spirometra) in Europe in the Molecular Era.

Sparganosis is a relatively neglected foodborne and waterborne disease caused by species of the tapeworm genus Spirometra, the global distribution of which has not been sufficiently recognized. Known mainly as a zoonosis of East Asia, its species are native to all inhabited continents including Europe. Spirometra has been reported from numerous wildlife species from 17 European countries, and a critical review confirmed 17 autochthonous and 8 imported human clinical cases. We present the first molecular evidence of the coincident presence of 2 species in Europe and review the current distribution to raise awareness of the parasite in this region. Spirometra erinaceieuropaei is restricted to Europe and Spirometra mansoni represents a lineage distributed mainly across Asia and Oceania that reaches Europe. The parasite is common in Eastern Europe and its distribution has potential to expand along with its invasive or migrating mammal hosts, spreading the risks of human infection.

Blastocystis isolates from a dog and their owners presenting with chronic diarrhoea. Dogs as reservoirs of Blastocystis: research in Poland and worldwide

Blastocystis cf. hominis is an unicellular protozoan parasite commonly found in the gastrointestinal tract of humans and animals. Blastocystis is characterized by high morphological and genetic diversity. Studies based on the analysis of Blastocystis spp. small subunit ribosomal RNA genes (SSU rDNA) have identified 26 subtypes (ST) so far, including at least 10 isolated from humans (STs 1-9 and ST12). In 2017, stool samples from a dog and its two owners living in Gdynia, Poland were examined; all three were suffering from chronic diarrhoea. In addition, 30 faecal samples were also examined from 30 dogs kept in one of Warsaw's hotels for animals. Stool specimens were analyzed using anaerobic cultivation at 37°C with a modified Jones' medium and molecular methods (PCR). Phylogenetic analysis using Bayesian inference was performed. Vacuolar forms of Blastocystis were identified in the stool samples of the dog and its owners; Blastocystis were not detected in any sample from the dogs living in the animal hotel. Based on the phylogenetic analysis, the obtained isolates were classified as subtype ST3 (for Owner 1) and subtype ST7 (for Owner 2 and the dog). To the best of our knowledge, the present study is the first to evaluate the presence of Blastocystis in canines in Poland, including domestic dogs.

Applying of bacteria Cellulosimicrobium sp. for cultivation protozoa of genus Acanthamoeba

The aim of this study was identification features of cultivation representatives of genus Acanthamoeba isolated from bentonite using Cellulosimicrobium sp. as a bacteria-feeders. Identification of isolated bacteria was conducted by morphological, cultural and molecular-genetic methods. The cultivation of free-living "bentonite" amoeba on the lawn of Cellulosimicrobium sp. have gained significant advantages than using Escherichia coli as a bacteriafeeders was shown. "Bentonite" amoeba form crateroid plaques, which fit to the quantitative characteristic materials which contains amoeba, during deep co-cultivation Acanthamoeba sp. and Cellulosimicrobium sp. on 1% glucose meet-peptone agar.

Morphological, physiological and genetic characteristics of protozoa of genus Acanthamoeba, isolated from different deposit of bentonite in Ukraine

The representatives of genus Acanthamoeba are widespread in the environment. The presence of freeliving Acanthamoeba sp. in such mineral deposits as bentonite was shown for the first time. Identification of isolated amoeba was conducted according to morphological features of trophozoites and cysts, as well as using sequencing of gene 18S RNA (amplifier GTSA.B1). The obtained data showed that isolated amoebae belong to the genotype T4 and II morphological group (cyst size <18 µm). For its growth, "bentonite" amoebae are intensively used bacteria of the genus Cellulosimicrobium sp. as a food substrate.

Onchocerca jakutensis ocular infection in Poland: a new vector-borne human health risk?

BACKGROUND: Zoonotic onchocerciasis is a vector-borne disease, which involves many animal species, including large ungulates, boars, dogs, and sporadically, humans. So far, 39 cases of zoonotic onchocerciasis have been reported worldwide, 30 of which have been found in the last 20 years. Onchocerca nematodes are transmitted to humans by blood-sucking vectors during a blood meal. The following species have been responsible for zoonotic infections: Onchocerca cervicalis, O. dewittei japonica, O. gutturosa, O. jakutensis and O. lupi. In humans, the worms have usually been found in the subcutaneous tissues where they form subcutaneous nodules, induce inflammation of musculature, or penetrate the eye. Thirteen ocular zoonotic onchocerciasis cases have been reported so far. In the eye, nematodes were localized in the subconjunctival space, anterior chamber and within the vitreous body. METHODS: In a 39-year-old male patient, a writhing worm in the vitreous body of the left eye was detected and surgically removed. Laboratory identification of the worm was based on macroscopic and molecular identification, based on sequencing of the cytochrome c oxidase subunit 1 gene (cox1). Phylogenetic analysis of the first 250 nucleotide sequences showing the highest levels of similarity with the present isolate in a BLAST analysis was performed. RESULTS: Here, we report the first case worldwide of human ocular infection with O. jakutensis, a natural parasite of red deer. By exploiting a PCR assay, we detected the sequence almost identical to O. jakutensis (GenBank: KT001213.1; positions 1-650) with a single mismatch G/A at position 622. The sequence reported in this paper was deposited in the GenBank database under the accession number MK491767. CONCLUSIONS: Our case together with the previous case reports indicate that zoonotic Onchocerca worms exhibit no tissue specificity and an eye infection has been described in over one third of human zoonotic onchocerciasis cases. In terms of the growing number of cases of zoonotic onchocerciasis in Europe, the USA and Japan, attention should be paid to the diagnosis of subcutaneous nodules and eye infestations.

Author Correction: Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hybrid de novo whole-genome assembly and annotation of the model tapeworm Hymenolepis diminuta.

Despite the use of Hymenolepis diminuta as a model organism in experimental parasitology, a full genome description has not yet been published. Here we present a hybrid de novo genome assembly based on complementary sequencing technologies and methods. The combination of Illumina paired-end, Illumina mate-pair and Oxford Nanopore Technology reads greatly improved the assembly of the H. diminuta genome. Our results indicate that the hybrid sequencing approach is the method of choice for obtaining high-quality data. The final genome assembly is 177 Mbp with contig N50 size of 75 kbp and a scaffold N50 size of 2.3 Mbp. We obtained one of the most complete cestode genome assemblies and annotated 15,169 potential protein-coding genes. The obtained data may help explain cestode gene function and better clarify the evolution of its gene families, and thus the adaptive features evolved during millennia of co-evolution with their hosts.

Dual infection of urinary tract with Enterocytozoon bieneusi and Encephalitozoon cuniculi in HIV/AIDS patients

Microsporidia are emerging pathogens which cause an opportunistic infections in immunocompromised patients, especially those with AIDS. Intestinal microsporidiosis is the most recognized infection, whereas urinary tract infections caused by microsporidia are rarely paid attention to either due to their subclinical course or diagnostic difficulties. In this report dual microsporidial infection of urinary tract, caused by Enterocytozoon bieneusi and Encephalitozoon cuniculi was described in HIV/AIDS patients under cART therapy. Since microsporidiosis can cause severe complications or even death in immunosuppressed patients, our results suggest that microsporidial infection should be included in routine investigation of HIV-positive patients, even asymptomatic.