This study was conducted with the aim of the molecular identification of the protozoan parasite Cryptosporidium spp. in calves in the early stage of their development on a dairy farm in Eastern Slovakia. Twenty-five Holstein and Holstein cross calves were included in the study and monitored from their birth to the fifth week of life (1–5 weeks). Fresh fecal samples were collected from the same group of calves each week, except during the fourth week, and with the exception of Sample 8. All samples were analyzed using the Ziehl–Neelsen staining method and coproantigen was tested using the ELISA test as the screening method. Using the ELISA method, the highest incidence of cryptosporidiosis was observed in the second week of life of the calves, while the antigen was detected in 21 (91.6%) calves. Using the Ziehl–Neelsen staining method, the highest incidence was also observed in the second week, with an incidence rate of 62.5%. Positive isolates confirmed by the ELISA test were molecularly characterized. The species and subtypes of Cryptosporidium in the positive isolates were identified using PCR and the sequence analysis of the small subunit of the ribosomal 18S RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. The sequence analysis of 29 isolates at the 18S rRNA loci confirmed the presence of two species—Cryptosporidium parvum and Cryptosporidium ryanae. Out of 29 isolates, 25 were assigned to the species C. parvum, with the gp60 locus identified as genotype IIaA17G1R1. Among the individual animal groups, calves are the most common reservoirs of the C. parvum zoonotic species. This disease has significant public health implications as contact with livestock and their feces and working with barn manure are major sources of infection, not only for other animals but also for humans.

The hepatitis B virus (HBV), belonging to the Hepadnaviridae family, is responsible for a global health concern still in the 21st century. The virus is divided into 10 genotypes, which differ in geographical distribution and in their effect on disease progression and transmission, susceptibility to mutations, and response to treatment. There are many methods for diagnostics of HBV and differentiating its genotypes. Various commercial kits based on real-time polymerase chain reaction (RT PCR) and hybridization available, as well as whole genome sequencing or the sequencing of only individual parts of the genomes. We compared a commercial kit AmpliSens HBV-genotype-FRT, based on RT PCR, with an adapted method of amplification of the surface genomic region combined with Sanger sequencing. In the examined samples we identified the A, B, C, D, and E genotypes. By PCR with Sanger sequencing, the genotypes were determined in all 103 samples, while by using the commercial kit we successfully genotyped only 95 samples, including combined genotypes, which we could not detect by sequencing.

Canine hookworms represent some of the most globally prevalent parasitic nematodes affecting dogs and pose a significant zoonotic risk to humans, in whom they can induce cutaneous larva migrans. Infection with these parasites may lead to blood loss, anaemia, and, in severe cases, mortality—particularly in young puppies. The present study reports a confirmed case of Ancylostoma caninum infection in a 15-month-old dog in the Slovak Republic. The infected dog presented with severe, persistent diarrhoea, and haematological examination revealed a decrease in erythrocyte and haemoglobin levels, as well as mild eosinophilia. Coprological examination confirmed the presence of nematodes belonging to the family of Ancylostomatidae. Following the initiation of anthelmintic therapy, adult individuals were recovered from the faeces. Based on distinct morphological characteristics, the parasites were presumptively attributed to the species A. caninum. Subsequent molecular analysis of the mitochondrial cytochrome c oxidase subunit 1 gene (COX1) and the ribosomal ITS regions definitively confirmed the species A. caninum. Our findings confirm that this is the first molecularly confirmed case of this species in Central Europe. This hookworm is predominantly found in warm and humid climatic regions. Its recent detection in Slovakia, a country characterised by a temperate climate, may suggest a northward expansion of its geographic range, potentially facilitated by ongoing climatic shifts associated with global climate change.

Culicoides biting midges (Diptera: Ceratopogonidae) are important vectors of avian haemosporidian parasites. Understanding their host preferences is crucial for elucidating transmission routes of vector-borne pathogens. In Slovakia, such knowledge is limited, particularly in forested wetlands. This study aimed to identify Culicoides species, their host preferences, and haemosporidian parasites in a wetland ecosystem at the Bird Ringing Station in Drienovec. Midges were collected in 2022 using UV light traps at two sites. In total, 2344 Culicoides individuals of 19 species were collected. Host blood was identified and DNA subsequently extracted from 36 engorged females, revealing feeding on three mammal and five bird species. The most frequently identified host was roe deer (Capreolus capreolus), predominantly fed upon by Culicoides obsoletus (Meigen 1818). Notably, avian haemosporidian DNA was detected for the first time in Slovakia in three Culicoides females. In two Culicoides alazanicus Dzhafarov 1961 individuals, DNA of Haemoproteus asymmetricus (TUPHI01) and Plasmodium matutinum (LINN1) was confirmed, both associated with avian blood from Turdus sp. One Culicoides festivipennis Kieffer 1914 female carried Haemoproteus tartakovskyi (HAWF1) and fed on Coccothraustes coccothraustes. These findings highlight the potential role of local Culicoides species in transmitting avian pathogens and underscore the importance of monitoring their ecology.

Ixodes ricinus is the most common and, epidemiologically speaking, the most important tick species in Slovakia, transmitting a wide range of zoonotic pathogens. The goal of the present study was to monitor selected tick-borne infectious agents in an urban environment in northern Slovakia where the conditions for their occurrence and survival are typically unfavourable. Ticks were collected by the flagging method during the period from March to November 2024 in the city of Žilina in five urban locations characterized by high human activity and suitable conditions for tick–host interactions. A total of 264 ticks of Ixodes ricinus were collected (67 females, 85 males, and 112 nymphs). A molecular analysis confirmed the presence of Borrelia spp. in 34.5% of samples, while the most frequently detected species was Borrelia afzelii. The other detected species included zoonotic piroplasms Babesia microti and Babesia venatorum (1.5%), as well as the bacteria Anaplasma spp. (2.65%) and Rickettsia spp. (0.4%). In four ticks (1.5%), the presence of coinfection caused by multiple pathogens was detected. These results confirm that urban ecosystems located in the northern regions of Slovakia also provide significant reservoirs of zoonotic pathogens and impose a potential risk for public health.