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Doppler ultrasonography is method suitable for observing and measuring the blood supply of many organs. Its principle is the change in the frequency of wave blood that moves with respect to the source of the wave (probe). In a testicular examination, the use of Doppler can also be very useful for detecting abnormalities or pathological conditions. The aim is just to characterize the flow in arteria testicularis in clinically healthy dogs aged 1-5 years. Twelve dogs were included in the study, of which 6 were rough collies weighing approximately 23 kg and 6 were standard dachshunds weighing approximately 9 kg. The pulsatility index (PI) and resistance index (RI) of a.testicularis, as well as the dimensions of the testicles - height and depth, were assessed by ultrasonographic examination using the ALOKA ProSound Alpha 6 ultrasonographic device (Hitachi-Aloka, Japan). The statistical evaluation of the results shows that the differences between the averages of both measurements of PI and RI indices are highly significant, so the weight of the individual had a high effect on the measured values of PI and RI indices. The depth of the testicles was also directly related to the increasing values of PI and RI indices and to the weight of the dogs, in contrast to their height, which did not show any statistical significance in this respect. The measured values and findings may in the future also serve as a basis for determining reference values in testicular ultrasonography according to the weight of dogs, which may be beneficial in clinical status assessment, diagnosis of various pathological conditions, or predicting spermatogenesis and fertility of the individuals.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco’s Modified Eagle’s Medium—32.55%) and the positive control (Dulbecco’s Modified Eagle’s Medium supplemented with FGF2, N2, B27, and EGF—82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

A 4-year-old intact female Standard Wirehaired Dachshund dog was referred with chief complaint of inguinal mass to the Small Animal Clinic at the University of Veterinary Medicine and Pharmacy in Košice, Slovakia. On physical examination, the bitch was healthy with no abnormalities other than having a non-painful and non-reducible soft tissue mass in the left inguinal region also involving the mammary gland. Ultrasonographic examination of the abdomen revealed a pregnant uterus and ultrasonography of the inguinal mass showed an organ with a lumen filled with anaechoic fluid. The diagnosis of a herniation was made and the owners decided for complete ovariohysterectomy. The left uterine horn had an incarcerated part of the inguinal hernia together with the ovary. Inside there was one macerated conceptus. The remaining part of the left uterine horn was located in the abdomen and had 2 macerated conceptuses. The bitch made an uneventful recovery from the surgery and anaesthesia with no postoperative complications and was sent home after the surgical procedure.

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.

Nosematosis is currently a frequently discussed honey bee disease caused by two types of Microsporidia: Nosema apis and Nosema ceranae. Nosematosis as an intestinal disease caused by these species is one of the main factors associated with the weakening and loss of hives, with none of the stressors acting in isolation and all having an important synergistic or additive effect on the occurrence of parasitic infection. The most important factors are exposure to pesticides and nutritional stress, both worsening the immune response. Honey bees Apis mellifera become more susceptible to parasites and subsequently the disease manifests itself. Choosing the right laboratory diagnostics is important to determine the prevalence of both species. Our review summarizes the most commonly used methodologies, especially polymerase chain reaction (PCR), which is a reliable method for detecting nosematosis, as well as for distinguishing between the two species causing the disease.

The most frequent canine perineal tumour is the perianal (gland) adenoma (also known as the circumanal adenoma or the hepatoid adenoma). Tumour markers are substances that are produced by the body in response to cancer, or may be produced by cancer itself. Carcinoembryonic antigen (CEA) and tissue polypeptide specific antigen (TPSA) are tumour antigens that are markers of carcinogenesis, with significantly elevated serum concentrations in the case of some types of tumours. In our study it has been proven that dogs with a perineal tumour (n = 35) had a mean value of CEA 0.69 ng·ml-1 compared to control dogs (n = 150) with 0.67 ng·ml-1 (P > 0.05). In TPSA dogs with perineal tumour, the mean value of TPSA was 15.86 IU·l-1 vs 13.92 IU·l-1 in the control dogs (P < 0.05). The results of the present study show that there is significant differences in TPSA between control dogs and group of dogs with a perineal tumour. Analysis of oncomarkers is a valuable tool for practitioners within small animal oncology, having an advantage over tissue biomarkers as the measurement procedure is non-invasive and shows dynamic changes of physiological and pathological states before clinical signs appear. Therefore, it may be used for an early detection of cancer.

Microsporidia are obligate intracellular pathogens that are currently considered to be most directly aligned with fungi. These fungal-related microbes cause infections in every major group of animals, both vertebrate and invertebrate, and more recently, because of AIDS, they have been identified as significant opportunistic parasites in man. The Microsporidia are ubiquitous parasites in the animal kingdom but, until recently, they have maintained relative anonymity because of the specialized nature of pathology researchers. Diagnosis of microsporidia infection from stool examination is possible and has replaced biopsy as the initial diagnostic procedure in many laboratories. These staining techniques can be difficult, however, due to the small size of the spores. The specific identification of microsporidian species has classically depended on ultrastructural examination. With the cloning of the rRNA genes from the human pathogenic microsporidia it has been possible to apply polymerase chain reaction (PCR) techniques for the diagnosis of microsporidial infection at the species and genotype level. The absence of genetic techniques for manipulating microsporidia and their complicated diagnosis hampered research. This study should provide basic insights into the development of diagnostics and the pitfalls of molecular identification of these ubiquitous intracellular pathogens that can be integrated into studies aimed at treating or controlling microsporidiosis.

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.

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.

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.

The aim of our study was to determine species and genotypes of Cryptosporidium in patients suffering from immunosuppressive illnesses, but also in immunocompetent patients suffering from diarrhoea. A total of 80 samples of faeces were collected from both immunosuppressed and immunocompetent patients. The immunosuppressed patients (65 samples) - 35 adult patients (group A) and 30 children (group B) were hospitalized at the Clinic of Oncohemathology. Samples from immunocompetent humans (15 samples, group C) were taken from patients with clinical signs of acute diarrhoea. With the use of molecular methods targeting the 60 kDa glycoprotein (GP60) gene region, we have identified multiple genotypes of Cryptosporidium. parvum and Cryptosporidium. hominis in immunocompromised, but also in immunocompetent individuals (C. hominis IbA10G2, IeA12G3T3; C. parvum IIaA10G1R1, IIaA11G2R1, IIaA12G2R1, IIaA13G1R1, IIaA14G1R1, IIaA14G2R1, IIaA17G1R1 and IIaA18G1R1). This is the first report of the occurrence of genotypes IIaA10G1R1, IIa12G2R1 and IIaA18G1R1 in human hosts.

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.

INTRODUCTION AND OBJECTIVES Chlamydia psittaci, an obligate intracellular bacterium, which is the etiologic agent of avian chlamydiosis in birds and ornithosis/psittacosis in humans, has been reported to be one of the most common pathogens found in feral pigeons worldwide, and thus constitutes a zoonotic risk. The aim of the study was to investigate pigeons in Slovakia living in areas in close proximity to humans for the presence of C. psittaci, using pharyngeal and cloacal swabs. MATERIAL AND METHODS 122 clinically healthy pigeons from different geographical regions of Slovakia were examined for the presence of C. psittaci. The adult pigeons of both genders were captured during the summer period in the urban centres of Slovakian towns. Each sample was examined by molecular method PCR, and in the case of positive result the identity of the obtained sequence was examined by a BLAST search. RESULTS Of the total number of 244 examined samples, 14 (5.7%) showed positivity for C. psittaci infection, 5 of which were from pharyngeal swabs (4.1%) and 9 from cloacal swabs (7.4%). A positive result was detected in 13 pigeons (10.7%). Phylogenetic analysis showed that all the positive samples are genetically very close to genotypes B and genotype E. CONCLUSION Phylogenetic examination of the 14 isolates of C. psittaci identified in the presented study, based on 23S rRNA gene sequence, revealed their close relationship with C. psittaci genotypes B and E. Both genotypes are predominantly prevalent in pigeons and both can be transmitted to humans. Therefore, it is necessary to perform screening examinations of animals and analyse the epidemiological factors affecting the way of transmission and circulation of pathogen.

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.

Cryptosporidium species C. parvum and C. hominis are a common cause of human diarrheal infections worldwide. In this study we attempted to detect these parasites in fish ponds and a water reservoirs in the Nitra region of Slovakia. We identified C. parvum genotype IIaA15G1R1 and genotype IIaA16G1R1 and C. hominis genotype IeA11G3T3 in these locations. Occurrence of the same genotype of C. hominis in two different locations indicates that this parasite is most likely present in the river that connects them. These results indicate a serious threat to public health of humans since these locations are used for fishing and for recreational activities.

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.

The topic of the study was to verify in vivo survival of in vitro cultured autologous and allogenous chondrocytes suspended in a fi brin glue Beriplast® or seeded on Collagen type I-Hyaluronan (Col type I-HYA) scaffolds for the regeneration of articular cartilage defects in rabbits. The study was carried out on 15 domestic rabbits randomly assigned to fi ve groups (n = 3 in each) with different treatments of artifi cially created chondral defects (ChD ́s). These defects were made in a non-load-bearing area of medial condyle of the distal femur, and were treated as follows: 1st and 3rd group: the ChD ́s were fi lled with autologous or allogenous chondrocytes seeded on Col type I-HYA scaffolds, respectively. The scaffolds were fi xed to the ChD ́s by fi brin glue Beriplast®; 2nd and 4th group: the ChD ́s were fi lled with a suspension of autologous or allogenous chondrocytes in fi brin glue Beriplast®, respectively, and they were immediately covered by unseeded Col type I-HYA scaffolds; Control group: the ChD ́s were left to heal spontaneously without any treatment. Macroscopical, histological and immunohistochemical analyses of the ChD ́s were performed 12 months after the treatment. In all treated groups, the chondrocytes were capable to proliferate and produce the cartilage extracellular matrix, including proteoglycans and type II collagen, as compared to the control “untreated” group. On the other hand, the production of hyaline-like cartilage tissue confi rmed that both therapeutic methods using autologous chondrocytes can be applied successfully for the treatment of chondral defects in rabbits. Cilj studije je bio da se verifi kuje in vivo preživljavanje in vitro kultivisanih autolognih i alogenih hondrocita suspendovanih u fi brinski Beriplast® lepak ili na kolagen Hyaluronan tip I (Col type I-HYA) osnovu radi regeneracije oštećenja zglobne hrskavice kod kunića. Ispitivanje je obavljeno na 15 domaćih kunića, odabranih slučajnim izborom koji su podeljeni u pet grupa (3 po grupi) kod kojih su veštački izazvana oštećenja hrskavice (ChD), tretirana na različite načine. Oštećenja su napravljena na površinama koje nisu noseće na medijalnom kondilusu distalnog femura, a tretman je podrazumevao: prva i treća grupa: ChD su punjene sa autolognim ili alogenim hondrocitima zasejanim na Col tip I-HYA osnovu. Osnove su fi ksirane na ChD pomoću fi brinskog lepka Beriplast®; druga i četvrta grupa: oštećenja su punjena suspenzijom autolognih ili alogenih hondrocita u fi brinskom Beriplast® lepku, a neposredno posle toga, prekriveni su sa nezasejanom Col tip I-HYA osnovom; kontrolna grupa: oštećenja su ostavljena da spontano zarastu, bez tretmana. Dvanaest meseci posle tretmana obavljene su makroskopske, histološke i imunohistološke analize promena. Kod svih životinja tretiranih grupa, hondrociti su bili sposobni da proliferišu i proizvode ekstracelularni matriks hrskavice, uključujući proteoglikane i II tip kolagena u poređenju sa kontrolnom “netretriranom” grupom. Sa druge strane, proizvodnja hrskavičavog tkiva nalik na hijalin potvrđuje da se oba terapijska metoda koji koriste autologne hondrocite mogu da uspešno primene u tretiranju defekata hrskavice kod kunića.

Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA and antisense oligodeoxynucleotides (ASON). The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%), retroviruses (344 clinical studies; 20.5%), unenveloped/plasmid DNA (304 clinical studies, 17.7%), adenoassociated viruses (75 clinical studies; 4.5%) and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.

[b]Introduction and objectives.[/b] [i]Chlamydia psittaci[/i], an obligate intracellular bacterium, which is the etiologic agent of avian chlamydiosis in birds and ornithosis/psittacosis in humans, has been reported to be one of the most common pathogens found in feral pigeons worldwide, and thus constitutes a zoonotic risk. The aim of the study was to investigate pigeons in Slovakia living in areas in close proximity to humans for the presence of C. psittaci, using pharyngeal and cloacal swabs. [b]Material and methods. [/b]122 clinically healthy pigeons from different geographical regions of Slovakia were examined for the presence of [i]C. psittaci[/i]. The adult pigeons of both genders were captured during the summer period in the urban centres of Slovakian towns. Each sample was examined by molecular method PCR, and in the case of positive result the identity of the obtained sequence was examined by a BLAST search. [b]Results.[/b] Of the total number of 244 examined samples, 14 (5.7%) showed positivity for [i]C. psittaci[/i] infection, 5 of which were from pharyngeal swabs (4.1%) and 9 from cloacal swabs (7.4%). A positive result was detected in 13 pigeons (10.7%). Phylogenetic analysis showed that all the positive samples are genetically very close to genotypes B and genotype E. [b]Conclusion.[/b] Phylogenetic examination of the 14 isolates of [i]C. psittaci[/i] identified in the presented study, based on 23S rRNA gene sequence, revealed their close relationship with [i]C. psittaci[/i] genotypes B and E. Both genotypes are predominantly prevalent in pigeons and both can be transmitted to humans. Therefore, it is necessary to perform screening examinations of animals and analyse the epidemiological factors affecting the way of transmission and circulation of pathogen.

The number of parasites followed the rapid growing of human population worldwide, not only in developing but also in developed countries. Many of them are diagnosed in children and adolescents. The occurrence of selected intestinal endoparasites in children coming from areas with low hygienic and socioeconomic status was studied. Out of 81 faecal samples examined, 46 (56.8 %) were positive for presence of intestinal parasites. From helminths, Ascaris lumbricoides was found to be the leading parasite (24.7 %), followed by Trichuris trichiura (17.3 %). Tapeworm Taenia spp. eggs were detected in 4.9 % of examined children. From protozoan parasites Cryptosporidium spp. was observed in 36 children (44.4 %) and Giardia intestinalis in 20 children (24.7 %). The occurrence of these epidemiologically low risky parasites in Roma children population suggests low hygienic standard in the Roma settlements.

Aim of the study: The purpose of this study was to find out the relationship between the phase of infection (acute or persistent) and the ability of quantitative PCR to detect DNA of Toxoplasma gondii in circulating leukocytes in blood. Methodology: Animal serum samples were examined (50 sheep, 47 dogs, 32 dairy cows, 91 wild boars and 36 rabbits) for the occurrence of IgM and IgG antibodies to T. gondii by ELISA. Uncoagulated blood samples from the same animals were examined for the detection of T. gondii DNA in circulating leukocytes by real-time PCR. Results: Only IgM antibodies, characteristic for acute infection, were detected in 45 of the 256 serum samples (17.6%). Only IgG antibodies, corresponding with chronic infection, were detected in 120 of the 256 samples (46.8%). In 91 of the 256 samples (35.5%) neither IgM or IgG were detected by ELISA. For real-time PCR, animals were divided into three groups based on the serological results: (group I — acute infection, group II — chronic infection, and group III — no infection). In group I, the presence of T. gondii DNA was detected in 9 out of 45 samples (20%), whereas in group II only 1 of 120 samples was positive for T. gondii DNA (0.8%). In group III, no DNA of T. gondii (0/91 samples) was detected by real-time PCR. Significance: The proof of DNA by real-time PCR in IgM positive samples was statistically significant in comparison to IgG positive samples (P

Microsporidiosis, cryptosporidiosis, blastocystosis, etc., of humans and animals are an intestinal diseases caused predominantly by infection with zoonotic species and genotypes of these pathogens. These diseasesare transmitted mainly via the faecal-oral route.Generally, these deseasesare occurs worldwide, but in places with poor sanitation and crowded living conditions is endemic and is associated with source of water and food supply, age, and socioeconomic status [1]. The diagnosis and genetic characterization of the main species and population variants of Microsporidia, Cryptosporidium and Blastocystis infecting humans andanimalsare central to the prevention, surveillance and control of these diseases, particularly as there is presently no cost effective chemotherapeutic regimen or vaccine available. In this Minireview we want to draw attention to the possibility of using HRM analysis for species and genotypic differentiation of these pathogens in clinical samples of humans and animals. This approach is well suited for the rapid screening of large numbers of Cryptosporidium oocyst, Blastocystis cyst and Microsporidia spores DNA samples and, although qualitative, is significantly less time-consuming to carry out than electrophoretic analysis. This method provides a useful tool for investigating the epidemiology and outbreaks of cryptosporidiosis, microsporidiosis and blastocystosis and could be applicable to identification of species of these pathogens.

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.

Several indicators ofimmune response were observed in immunocompetent mice of the ICR line and those suppressed by dexamethasone upon their experimental infection with the microsporidia of Encephalitozoon cuniculi. The mice were infected by one-shot intraperitoneal administration of 5×107 pathogenic spores. On Days 7, 14, 28 and 42 after infection, peripheral blood leukocyte phagocytic activity was determined and compared, including phagocytic index and the blastogenic response in spleen cells to mitogenic activation by concanavalin A and phytohaemagglutinin. The results point to the fact that E. cuniculi itself can cause a significant decrease in phagocytic activity of phagocytic leukocytes in the early stages of infection as well as a remarkable decrease in the proliferative response of spleen cells to T-cellular mitogens.

Wild animals can be involved in epidemiology of many important diseases and often act as reservoirs of pathogens which cause disease in domestic animals and humans. This paper aims the role of red fox (Vulpes vulpes) and brown bear (Ursus arctos) in the circulation of coccidian parasites from the genus Cryptosporidium. Cryptosporidiosis is known as an important enteric pathogen, clinical symptoms in particular in immune-compromised individuals range from mild to severe diarrhoea and dehydration, which could be fatal. Fecal samples from 62 red foxes shot during September 2010 to February 2011 and 63 brown bears collected during June 2010 to March 2011 in central and eastern Slovakia were examined for the qualitative determination of Cryptosporidium spp. antigens in faeces by sandwich ELISA kit. Overall, 38.7% (24/62) of faecal samples of red foxes and 55.6% (35/63) of faecal samples of brown bear were positive. Our preliminary results emphasize prevalence of Cryptosporidium spp. amongst brown bears and red foxes in Slovakia and highlight the potential risk for transmission of cryptosporidiosis to humans using the countryside for professional or recreational purposes.

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

Microsporidia are identified as ubiquitous organisms of almost every animal group and are now recognized as emerging opportunistic pathogens of human. The risk factors include immunodeficiency, lack of sanitation, and exposure to contaminated water and infected animals. In Slovakia, the places with an increased risk of infection due to the presence of risk factors and routes of transmission are represented by Roma settlements. Therefore, the aim of this work was to study the occurrence of Encephalitozoon spp. and E. bieneusi in children living in Roma settlements.Stool samples were examined of 72 clinically healthy children coming from a group of the non-integrated Roma minority for the presence of microsporidia Encephalitozoon spp. and E. bieneusi. Microsporidian spores were detected by standard Rylux D, staining and by PCR and DNA sequencing.Of the total number of 72 stool smears examined, 22 were positive, which represented 30.6%. By the Real Time PCR, E. bieneusi was detected in 3 samples (4.2 %) and E. cuniculi in 19 samples (26.4 %). By comparing the sequences with sequences in the GenBank, E. cuniculi genotype I (Accession No. AJ005581.1) and E. bieneusi genotype A (Accession No. AF101197.1).Microsporidia, as newly emerging pathogens of humans and animals, are characterised by the production of spores which are environmentally resistant. Diseases caused by them have a cosmopolitan occurrence. Although E. bieneusi and E. cuniculi belong to the most frequently diagnosed species of microsporidia in humans, in Slovakia, this is the first confirmed evidence of E. bieneusi genotype A, as well as E. cuniculi genotype I in humans by the molecular method.

Cryptosporidiosis is considered to be a widespread world zoonosis. The occurrence of Cryptosporidium species was investigated in Roma children in a district of Eastern Slovakia and, at the same time, also in children of non-Roma parents. In total, 103 children (54 boys and 49 girls) between 0 and 14 years of age were involved in this study. Fifty-three were Roma children and 50 children represented a non-Roma control group. Fecal samples were examined: immunologically [enzyme-linked immunosorbent assay (ELISA) test to prove antigen in the feces] and by molecular analysis [nested polymerase chain reaction (PCR)]. After the sequencing of the PCR, the products were identified as species of Cryptosporidium muris. Based on the results, the relative risk (RR) of the Cryptosporidium infection occurrence was calculated and we came to the conclusion that the risk of Cryptosporidium infection was almost 12 times higher in the Roma children compared to the non-Roma children.

A total of 47 avian faecal samples of wild waterfowl (great cormorant — Phalacrocorax carbo, great crested grebe — Podiceps cristatus, white stork — Ciconia ciconia) trapped in the eastern Slovakia were screened for the presence of human pathogenic microsporidia by microscopy and real-time SYBR Green PCR method using species primers and sequenced. Microscopic analysis showed presence in 32 samples (29 cormorants, 3 dippers). Microsporidial DNA (Encephalitozoon cuniculi genotype I) was identified in 19 faeces samples (40.4%) namely cormorants in 17 out of 40, one dipper of 5 and a stork out of 2. The present work describes three new host species of the bird population in microsporidium Encephalitozoon cuniculi genotype I which confirms the theory of low specificity of this species.

Parasitic pathogenic protozoa largely parasitize intracellularly, the course of these infections is acute, often cause the death. On the other hand, they can progress subclinically. The latent respectively chronic stage can follow the acute form and infections can persist throughout the whole life of the host. The course of the disease mostly depends also on the pathological agents. They stimulate the innate and adaptive immune response of the host. In mostly protozoan infections the immune response is not so sufficiently effective for a complete destruction of the parasite. This situation ensures the survival of the parasite and it is the characteristic feature for mostly protozoan infections.

The microsporidia are emerging agents of infectious disease in both immunocompromised and immunocompetent mammals. Recently, there has been an increased interest in studying the immunobiology of microsporidiosis. This paper discusses the humoral and cell-mediated immune responses to Encephalitozoon spp. The T-cell-mediated responses appear to be most important in conferring resistance. This has become evident by the lethal effects of microsporidiosis in T-cell-deficient hosts. However, much still needs to be learned about the immunobiology of microsporidiosis regarding the specific T-cell responses and the cytokines that provide protective immunity and facilitate the macrophage-mediated killing of microsporidia. Such information will become important in developing immunotherapeutic strategies to control microsporidiosis in the future.

Recently, the pathogenic species of microsporidia of the genus Encephalitozoon have been detected increasingly, also in representatives of the Aves class. Our study presents laboratory proof of Encephalitozoon cuniculi (E. cuniculi) genotype II in a new host, gyrfalcon (Falco rusticolus), with suspect microsporidiosis. E. cuniculi is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. Characterization of the internal transcribed spacer of the rRNA gene has identified three genotypes of E. cuniculi based on the number of 5′-GTTT-3′ repeats present: a genotype I from rabbits and mice, containing three repeats; a genotype II from mice and dogs, containing two repeats; and a genotype III from dogs and fox, containing four repeats. Samples of faeces from 30 gyrfalcons were examined for the presence of microsporidia spores, using microscopical, molecular methods and sequencing. Microscopic analysis showed presence of brightly fluorescing oval shapes of size 1.5 × 3 μm, characteristic of the strain Microsporidia in five samples. The PCR method, using species non-specific (530F/580R) and species-specific (ECUNR/ECUNF) primers, proved the presence of E. cuniculi spores in two samples. After sequencing were confirmed, E. cuniculi genotype II which implies new host species for this parasite.

The paper presents the results of examination of 32 domestically bred rabbits, the breed Nederland Dwarf of Oryctolagus cuniculus, for the presence of Encephalitozoon cuniculi microsporidian species. The results of serological tests for E. cuniculi in 32 rabbits are reviewed along with other follow-up studies of clinical cases. Blood samples were taken from 7 asymptomatic rabbits and 25 rabbits showing neurological and ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, 5 out of 7 rabbits were seropositive (71%). 16 rabbits with clinical diseases showed neurological sings, including torticollis, circus-like movements, loss of weight; 6 of them also showed ataxia, anorexia, asthenia of hind-limbs and 3 showed ocular signs. All 25 rabbits were seropositive. The spores of E. cuniculi were isolated from the faecal samples or kidneys and brain of an animal and subsequently were used for DNA isolation and PCR analysis.

Encephalitozoon intestinalis infection of sows is reported from a pig farm in Slovakia. Spores were detected by direct microscopic visualisation in the faeces of 25 out of 27 sows (92.6%). This finding was also supported serologically by the presence of specific anti-E.intestinalis antibodies and by a species-specific polymerase chain reaction (PCR). This is the first report on E. intestinalis infection of swine in Europe.

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.