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.

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.

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 Košice 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.

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.

Despite the fact that Cryptosporidium spp. is a parasite which commonly causes diarrhea, it still receives little attention. In our experiment, we focused on comparing the biological (N. davidi shrimp) and physical (zeolite with different thicknesses) possibility of filtering cryptosporidia from a small volume of water, which could contribute to increasing the catchability of this parasite. We monitored the ability to capture oocysts of the parasite Cryptosporidium parvum, genotype IIaA11G2R1, found in water samples. We infected drinking water with feces with a known number of cryptosporidial oocysts. One gram of sample contained ±28 oocysts. We filtered eight water samples with different concentrations of oocysts (0.1–2 g of infected stool per 15 L of water) using zeolite with a particle thickness of 0.2–0.6 mm and 0–0.3 mm. This was followed by purification, centrifugation and isolation utilizing the isolation kit AmpliSens® DNA-sorb-B, which is intended for stool. In total, 120 shrimp were divided into four aquariums (A, B, C, n = 30) including the control (K), while drinking water with the same parameters was infected with different concentrations of oocysts (A: 2.5 g, B: 2 g, C: 1 g of infected stool per 15 L of water). We took 10 individual shrimp and processed them in three time intervals (6 h, 12 h and 24 h). We processed them whole, and we isolated the DNA utilizing the isolation kit AmpliSens® DNA-sorb-AM, which is intended for tissues. Detection was carried out by molecular methods, namely the Nested PCR targeting of the region of the GP60 gene (60 kD glycoprotein). Gel electrophoresis showed the presence of C. parvum in seven zeolite-filtered water samples, and the parasite was not found in the water sample with the lowest number of oocysts filtered through the smaller-particle zeolite. There were 67 C. parvum-positive shrimp. Whereas the most positive shrimp were identified at 12 h of sampling, the least were identified at the 24 h mark. No shrimp positive for C. parvum was found in the control group. By sequencing, we confirmed the presence of C. parvum, genotype IIaA11G2R1, in all positive samples. We thus proved that the filtration capabilities of zeolite and N. davidi can be used for the rapid diagnosis of the presence of protozoa in a small amount of studied water.