Detection of Blastocystis spp., Cryptosporidium spp. and Encephalitozoon spp. among wild animals from Eastern Slovakia.

The aim of this study was to draw attention to the risk of transmission of Encephalitozoon, Cryptosporidium and Blastocystis infection due to high animal migration and to point out that even wild animals can be a source of many zoonotic diseases. Encephalitozoon cuniculi, Cryptosporidium spp. and Blastocystis spp. are frequent microscopic organisms that parasitise humans, domestic and wild animals. Two hundred and fifty-five faecal specimens were collected from wild boars, badgers, wolves, bears, foxes and deer from 15 locations in Slovakia. Sequencing of positive PCR products and subsequent sequence comparison with GenBank sequences identified Blastocystis spp. in five wild boars. The ST 5 (n = 4) and ST 10 (n = 1) subtypes were determined by genotyping. We identified Encephalitozoon cuniculi in five wild boars, and genotype II (n = 5) was determined on the basis of ITS repeat sequences. Cryptosporidium scrofarum was sequenced in wolves (n = 4) and wild boars (n = 1), while Cryptosporidium suis only in wild boars (n = 2). None of the wild boars had a mixed infection.

First report of Blastocystis spp. subtypes in ZOO animals in Slovakia, Central Europe.

Blastocystis spp. has been reported in wildlife, domestic animals and animals housed in ZOO. To-date, 17 genetically diverse lines have been reported in mammals and birds (designated ST) based on differences in the SSU rRNA. In this study, faeces samples were collected from 24 ZOO animals with clinical signs suggestive of gastrointestinal disease in Kosice ZOO, Slovakia. After DNA isolation, PCR was conducted to amplify the SSU region of DNA of Blastocystis species. Forward primer- Blast F and reverse primer- Blast R were used in the reaction. From 25 faeces samples, Blastocystis spp. was detected in 5 animals (3 mammals, 2 birds), with a prevalence of 20%. Subsequent molecular analyses identified the ST 5 (n = 3), ST 7 (n = 1), and ST 12 (n = 1) subtypes, where the ST 5 subtype was identified in the mammalian group and birds, and the ST 7 and ST 12 subtypes were identified only in mammals. Based on these findings, focusing on ZOO animals as a potential source of infection for humans is highly recommended.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli.

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

First detection of Blastocystis sp. in pigs in Slovakia and in Europe.

Blastocystis sp. is a single-cell microorganism occurring in the gastrointestinal tract of humans and various animals and is distributed worldwide. Blastocystis exhibits extensive genetic diversity of 28 subtypes (STs) based on the small subunit ribosomal RNA (SSU rRNA) gene. In this study, the genetic diversity and zoonotic potential of Blastocystis were evaluated using pig faecal samples from two farms in Slovakia. Blastocystis spp. were detected in pigs intended for distribution and consumption. ST 5 subtype was identified in all positive samples and age categories with a prevalence of 12%. However, the prevalence on one of the farms was up to 28.6%. This is the first study of Blastocystis in pigs carried out in Slovakia. Although a number of samples obtained was small, the identified subtype of ST5 Blastocystis sp. occurs in humans and animals. It may have zoonotic potential and therefore may be a risk factor due to the close contact between humans and pigs on the breeding farms.

Molecular characterization of new genotypes Enterocytozoon bieneusi in Slovakia.

Enterocytozoon bieneusi is characterized as a ubiquitous intestinal parasite with a wide genetic diversity, and it is capable of infecting a diverse range of hosts all around the world. Since information about the genotype diversity of E. bieneusi in pigs, calves, sheep and goats in Slovakia is very limited, we examined three farms where we mapped the occurrence of E. bieneusi and its genotypes, thus contributing to the information about geographic diversity of this pathogen worldwide. In this study we used PCR methods to examine 253 fecal samples from pigs, calves, sheep and goats with suspected microsporidiosis. Real time PCR was used to identify genotypes by amplification of SSU region and ITS region. After analysis we detected presence of E. bieneusi (7) and Microsporidia sp. (6) in 13 samples. The analysis of nucleotide sequences of ITS region of E. bieneusi shows, that the positive isolates belonged to 5 genotypes, including two known genotypes (I, F) and three new genotypes diagnosed in pigs, named SVK-S1, SVK-S2 and SVK-S3. Phylogenetic analysis showed that these novel genotypes identified in present study belong to group 1, which previously has been described as a zoonotic group. Genotype I was detected in two calves and genotype F was detected in two pigs.

Screening of opportunistic Encephalitozoon intestinalis and Enterocytozoon bieneusi in immunocompromised patients in Slovakia.

OBJECTIVE: In recent years new infectious diseases, i.e. emerging or re-emerging diseases, have been coming to the forefront. Currently, microsporidia, considered to be a major cause of emerging and opportunistic infections particularly in immunocompromised individuals, are also included in this group. Therefore, the aim of our study was to map the prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi infection in a group of patients and to compare it with the occurrence of specific antigens in immunocompetent people. METHODS: Detection of spores of both pathogens in faecal samples was performed by an immunofluorescence test using species-specific monoclonal antibodies. RESULTS: Positivity to E. intestinalis in 91 examined immunosuppressed patients reached 33% (30/91), while only 4.3% (3/70) of the control group samples were found to be positive (relative risk 7.7, p < 0.001). In case of E. bieneusi 14.3% (13/91) of immunocompromised patients were positive, as were 5.7% (4/70) of people from the control group (relative risk 2.5, p = 0.095). CONCLUSION: In case of development of any opportunistic infection, the infection is detected and removed in most cases at an early stage. The incidence of clinically manifested microsporidiosis in patients with immunodeficiency is rare as they are under constant medical supervision. However, we must not forget about opportunistic infections, and in case of any non-specific symptoms it is necessary to exclude or confirm the diagnosis for immediate treatment.

Chlamydiosis in farmed chickens in Slovakia and zoonotic risk for humans.

INTRODUCTION: Chlamydia psittaci is an obligate intracellular Gram-negative bacterium causing respiratory disease (chlamydiosis) or asymptomatic carriage in poultry. In humans, it is a zoonotic agent of ornithosis/psittacosis. Due to low awareness of the disease and variable clinical presentation, psittacosis is often remains unrecognised as such by general practitioners. Zoonotic transfer occurs through inhalation of contaminated aerosols, and originates from feathers, faecal material and respiratory tract exudates. OBJECTIVE: The aim of this study was to investigate chickens for the presence of Chlamydia sp. from pharyngeal and cloacal swabs and review the zoonotic risk for humans. MATERIAL AND METHODS: 138 clinically healthy chickens from farms in Slovakia were examined for the presence of Chlamydia sp. The age of the chickens was 6 months. Two different samples were used - pharyngeal swabs and cloacal swabs. Each sample was examined by the molecular PCR method, and in the case of a positive result the identity of the obtained sequences was examined by a BLAST search. RESULTS: Of the total number of 276 examined samples from 138 chickens, 19 (6.9%) showed positivity for C. psittaci infection, 12 (8.7%) which were positive from pharyngeal swabs and 7 (5.1%) from cloacal swabs. None of the chickens were positive in both samples. Phylogenetic examination of the 19 isolates identified in the study, based on the 23S rRNA gene sequence, revealed that the isolates obtained were identical with C. psittaci, and genetically very close to genotypes B and genotype E. CONCLUSIONS: C. psittaci infections are apparently emerging in chickens. Chicken-processing plant employees should be considered a risk group for human psittacosis. There is a need for higher awareness and for efficient risk assessment and management.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals.

The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN®Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.

Rodents as a reservoir of infection caused by multiple zoonotic species/genotypes of C. parvum, C. hominis, C. suis, C. scrofarum, and the first evidence of C. muskrat genotypes I and II of rodents in Europe.

Cryptosporidium spp. is an important causative agent of intestinal parasitoses-induced diarrhoea in humans and animals worldwide. Rodents (small mammals), the main reservoir of infections, are globally expanded and overpopulated, which increases the risk of transfer of human and zoonotic pathogens from the genus Cryptosporidium. In this study, Cryptosporidium was detected in wild immunocompetent asymptomatic small mammals. Altogether 262 fecal samples were collected from five areas in Eastern Slovakia from four different rodent species (Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Rattus norvegicus), eight samples originated from two insectivore species (Sorex araneus, Crocidura suaveolens), and two sample from a carnivore Mustela nivalis. The samples were examined using a method modified in our laboratory, based on the use of specific primers on a small subunit rRNA (18S rRNA) gene for species identification, and amplification of GP60 gene coding 60-kDa glycoprotein for genotype determination. The following species were identified: Cryptosporidium parvum (n=15), genotypes IIaA18G3R1 (n=11; KU311673), IIaA10G1R1 (n=1; KU311670), IIcA5G3a (n=1; KU311669), IIiA10 (n=2; KU311672); Cryptosporidium suis (n=4; KU311671); Cryptosporidium scrofarum (n=28); Cryptosporidium environment sp. (n=12; KU311677); Cryptosporidium muskrat genotype I (n=3; KU311675); Cryptosporidium muskrat genotype II (n=3; KU311676). From one of the rodent, the species Cryptosporidium hominis genotype IbA10G2 (KU311668) was identified for the first time. The results of this study indicate low host specificity of the detected Cryptosporidium species and imply the importance of free-living small mammals in urban and suburban habitats as a potential source of human cryptosporidiosis.

Detection and identification of six Cryptospordium species in livestock in Slovakia by amplification of SSU and GP60 genes with the use of PCR analysis.

INTRODUCTION: In this study we examined 200 faecal samples from pigs and calves with suspected cryptosporidiosis were examined by the PCR methods: nested PCR for amplification of SSU region; nested PCR for amplification of GP60 region; and with restriction analysis of DNA (PCR-RFLP). The sequencing identified the following species: Cryptosporidium muris (2), Cryptosporidium andersoni (1), Cryptosporidium bovis (4), Cryptosporidium suis (2), Cryptosporidium scrofarum (10), mixed infection caused by C. scrofarum and C. muris (1),and Cryptosporidium parvum (10) genotype A subtype IIaA17G2R1. RESULTS AND CONCLUSIONS: The findings suggest that livestock can be an important source of zoonotic species or genotypes of Cryptosporidium, which may adversely affect the public health of human populations. This is the first time in our country that the Cryptosporidium species has been identified in livestock in Slovakia. The identification and genotyping of this pathogen in Slovakia, completes the epidemiological situation in Europe for Cryptosporidum species.

Molecular characterization and first report of Cryptosporidium genotypes in human population in the Slovak Republic.

In our study, we examined 91 fecal samples from five different groups of people containing HIV patients, hemodialysis patients, kidney transplant recipients, immunocompetent humans without clinical signs, and humans with suspected cryptosporidiosis. The purpose of our study was to determine species and genotype composition of representatives of Cryptosporidium spp. using PCR analysis of small subunit ribosomal RNA gene and 60-kDa glycoprotein gene and examine their phylogenetic relationship. In HIV-positive/AIDS-infected group of patients and in hemodialysis patients, no presence of Cryptosporidium species was detected. In two kidney transplant recipients, we detected species/genotypes Cryptosporidium parvum IIaA13G1T1R1 (KT355488) and Cryptosporidium hominis IaA11G2R8 (KT355489) and in two immunocompetent patients with clinical symptoms, we identified Cryptosporidium muris and C. hominis IbA10G2T1 (KT355490). In the group of healthy immunocompetent individuals without clinical signs, we identified species/genotype C. hominis IbA11G2 (KT355491) in one sample.

First report of Enterocytozoon bieneusi and Encephalitozoon intestinalis infection of wild mice in Slovakia.

Increased risk of zoonotic transmission of the potential human pathogenic species Enterocytozoon bieneusi, Encephalitozoon intestinalis and Encephalitozoon cuniculi was detected in wild immunocompetent mice (Mus musculus musculus; n=280). Analysis was conducted with the use of PMP1/PMP2 primers and SYBR Green RT-PCR. Using Real Time PCR and comparing the sequences with sequences in the GenBank, E. bieneusi was detected in 3 samples (1.07 %), E. cuniculi in 1 sample (0.35 %) and E. intestinalis in 1 sample (0.35 %). The results of this report document the low host specificity of detected microsporidia species, and imply the importance of synanthropic rodents as a potential source of human microsporidial infection.

Serological screening of selected microsporidia in HPV-positive women.

Microsporidiosis is considered to be emerging opportunistic infection in immunocompromised individuals worldwide. The aim of this study was to identify the specific serum antibodies to intestinal microsporidia Encephalitozoon cuniculi and Encephalitozoon intestinalis in women with Human Papillomavirus HPV and without HPV by the indirect immunofluorescence (IFA). From total number of 669 examined women, 225 were HPV positive and 444 women HPV negative. Overall the study comprised of 10.8% women with positive result for presence of E. cuniculi antibodies. In group 1 (HPV-positive women) it was more than 28% and in group 2 (HPV-negative women) it was less than 2% (p<0.001). E. intestinalis infection was found in total of 4.48% women, in group 1 it was present in less than 6% and in group 2 in less than 4% of women.