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A permanently high virulence was found in tachyzoites of T. gondii K24 after serial passage in mice (90 passages during 324 days). Virulence tests revealed that a single tachyzoite of the 50th passage represented LD100 for mice. Analysis of genotype of K24 isolate was done by PCR/RFLP with ROP1/DdeI, SAG1/DdeI, 850/RsaI and IGS/RsaI and by RFLP/DNA with TGR 1E sequence and PstI enzyme. K24 isolate had an atypical genotype, with an association of type II (for ROP1, SAG1 genes and TGR1 E sequence) and type I (for 850 gene) alleles, and a new pattern observed for IGS. All tested PCR/RFLP did not change through 2, 10, 20, 28, 40, 50, 60, 70, 81 and 90 tested passages. In RFLP/DNA with PstI enzyme and TGR1 E probe, K24 isolate produced a pattern with seven fragments of the size ranging from one to 23 kb and did not change through 7, 56, 70 and 83 tested passages. K24 T. gondii isolate is a hybrid and has the virulence of lineage I isolates.

Abstract Background Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. Results We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. Conclusion Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.

Purpose: Parasites of genus Encephalitozoon are well known pathogens of domestic animals however less attention was paid to its spread among wildlife that can play an important role of reservoir of infection. The aim of the study was to conduct molecular detection and genotype characterization of Encephalitozoon spp. in wild small mammals trapped in localities both near to and at a large distance from residential areas., Methods: In total, 300 wild small mammals (274 Rodentia and 26 Eulipotyphla) were trapped in 41 localities of the Czech Republic and tested by nested PCR for Encephalitozoon spp., Results: The DNA of Encephalitozoon spp. was proved in tissues (brain or liver) of 11% (32/300) of animals. There was a statistically significant difference (p < 0.001) in positivity among animal species with the most infected species Micromys minutus (50%, 4/8) and Myodes glareolus (17%, 9/53). There was also statistically significant difference (p < 0.001) between localities with the higher positivity (29%, 12/42) in localities near to residential areas, compared to localities with a large distance from residential areas (8%, 20/258). Sex and age of wild small mammals did not have effect on their positivity. Genotyping analysis revealed E. cuniculi genotype II in 22 samples and E. hellem genotype 1 A in one sample., Conclusion: This study brings new information on the molecular characterization of Encephalitozoon spp. isolated from wild small mammals trapped in two different areas (localities in near to residential areas and localities with a large distance from residential areas)., Competing Interests: Declarations. Ethical Approval Statement: The trapping of animals and the sampling were conducted according to the experimental projects approved by the Administration of the Poodří Protected Landscape Area, by the Administration of the Moravian Karst, and by the Ministry of the Environment of the Czech Republic (projects no. 41/2015, no. 8/2016, and no. 42/2015). Competing Interests: The authors declare no competing interests. Conflict of Interest: None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper., (© 2024. The Author(s).)

Gene therapy is a focus of interest in both human and veterinary medicine, especially in recent years due to the potential applications of CRISPR/Cas9 technology. Another relatively new approach is that of epigenetic therapy, which involves an intervention based on epigenetic marks, including DNA methylation, histone post-translational modifications, and post-transcription modifications of distinct RNAs. The epigenome results from enzymatic reactions, which regulate gene expression without altering DNA sequences. In contrast to conventional CRISP/Cas9 techniques, the recently established methodology of epigenetic editing mediated by the CRISPR/dCas9 system is designed to target specific genes without causing DNA breaks. Both natural epigenetic processes and epigenetic editing regulate gene expression and thereby contribute to maintaining the balance between physiological functions and pathophysiological states. From this perspective, knowledge of specific epigenetic marks has immense potential in both human and veterinary medicine. For instance, the use of epigenetic drugs (chemical compounds with therapeutic potential affecting the epigenome) seems to be promising for the treatment of cancer, metabolic, and infectious diseases. Also, there is evidence that an epigenetic diet (nutrition-like factors affecting epigenome) should be considered as part of a healthy lifestyle and could contribute to the prevention of pathophysiological processes. In summary, epigenetic-based approaches in human and veterinary medicine have increasing significance in targeting aberrant gene expression associated with various diseases. In this case, CRISPR/dCas9, epigenetic targeting, and some epigenetic nutrition factors could contribute to reversing an abnormal epigenetic landscape to a healthy physiological state., Competing Interests: I declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Chemical modifications in mRNAs, tRNAs, rRNAs, and non-coding RNAs stabilize these nucleic acids and regulate their function. In addition to regulating the translation of genetic information from mRNA to proteins, it has been revealed that modifications in RNAs regulate repair processes in the genome., Methods: Using local laser microirradiation, confocal microscopy, dot blots, and mass spectrometry we studied the role of N7-methylguanosine (m7G), which is co-transcriptionally installed in RNA., Results: Here, we show that after UVC and UVA irradiation, the level of m7G RNA is increased initially in the cytoplasm, and after local laser microirradiation, m7G RNA is highly abundant in UVA-damaged chromatin. This process is poly(ADP-ribose) polymerase (PARP)-dependent, but not accompanied by changes in the level of m7G-writers, including methyltransferases RNMT, METTL1, and WBSCR22. We also observed that METTL1 deficiency does not affect the recruitment of m7G RNA to microirradiated chromatin. Analyzing the levels of mRNA, let-7e, and miR-203a in both the cytoplasm and the cell nucleus, we revealed that UVC irradiation changed the level of mRNA, and significantly increased the pool of both let-7e and miR-203a, which correlated with radiation-induced m7G RNA increase in the cytoplasm., Conclusions: Irradiation by UV light increases the m7G RNA pool in the cytoplasm and in the microirradiated genome. Thus, epigenetically modified RNAslikely contribute to DNA damage responses or m7G signals the presence of RNA damage., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Introduction: There have only been a few molecular studies conducted on the detection of T. gondii in tissues of carnivores in South Africa, with no data on the genetic diversity of this parasite. That is why the aim of this study was to detect and genotype T. gondii DNA in tissues of selected wild and domestic carnivores in South Africa., Methods: Samples were collected from 80 animals of 20 species (mainly road-killed) in the four provinces of Limpopo (n=57), Mpumalanga (n=21), Gauteng (n=1) and Free State (n=1) during the period 2014-2018. Samples of brain (n=31), heart (n=4), liver (n=40), spleen (n=2) and lung (n=3) were used to detect T. gondii by real-time PCR targeting a 529 bp repeating fragment of T. gondii DNA. Samples that were positive in real-time PCR were genotyped using 15 microsatellite markers., Results: T. gondii DNA was detected in 4 (5 %) samples: in the brain from a Black-backed Jackal ( Canis mesomelas ), in the liver from a African Wildcat ( Felis silvestris lybica ) and in the liver and heart of two Rusty-spotted Genets ( Genetta maculata ) respectively. The DNA sample from Black-backed Jackal was genotyped and characterized as belonging to the type Africa 4 lineage (equivalent to RFLP genotype ToxoDB#20), that is a widespread lineage in Africa., Discussion: This is the first genetic characterization of T. gondii isolated from a wild carnivore on the African continent and the first report of T. gondii in Black-backed Jackal. The Africa 4 lineage was also confirmed in the region of Southern Africa for the first time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Račka, Bártová, Hamidović, Plault, Kočišová, Camacho, Mercier and Halajian.)

Lyme disease, caused by some strains of bacterial spirochetes Borrelia burgdorferi sensu lato ( Bb sl), affects humans but also domestic animals including horses. The primary pathogens in horses in Europe are B. afzelii , B. garinii and B. burgdorferi sensu stricto. To our knowledge, there are no data available on the seropositivity of B. burgdorferi s.l. in horses from the Czech Republic. In this country, horses are mainly used for sport, breeding, and recreational riding in areas where vectors of B. burgdorferi s.l. are present, which is why they are frequently at risk of infection. The aim of the study was to detect anti-borrelia IgM and IgG antibodies in clinically healthy and sick horses from the Czech Republic and to evaluate the risk factors of infection. In total, sera of 262 horses (247 clinically healthy horses and 15 horses hospitalized due to symptoms of encephalitis/meningoencephalitis) were examined by an indirect sandwich enzyme-linked immunosorbent assay. Positivity of B. burgdorferi was 27% (66/247) in clinically healthy horses (21% IgM, 7% IgG and 3% IgM + IgG antibodies) and 20% (3/15) in horses with clinical signs (20% IgM, 7% IgG and 7% IgM + IgG). In the clinically healthy horses, positivity statistically differed ( p ≤ 0.05) only in Pony and Warmblood breeds, being the most affected at 32% and 30%, respectively, while other characteristics (sex, age, usage and localities) had no effect on positivity. This is the first survey of antibodies to B. burgdorferi s.l. in Czech horses showing that horses are exposed to ticks infected with B. burgdorferi s.l. This should be taken into account when making differential diagnoses in patients with non-specific symptoms to start with adequate therapy.

RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs., (© 2023. The Author(s).)

Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: Toxoplasma gondii , Neospora caninum and microsporidia Encephalitozoon spp. In total, brain and small intestine tissues of 100 bats (52 Myotis myotis , 43 Nyctalus noctula and 5 Vespertilio murinus ) were used for the DNA isolation and PCR detection of the abovementioned agents. Toxoplasma gondii DNA was detected by real-time PCR in 1% of bats (in one male of M. myotis ), while all bats were negative for N. caninum DNA. Encephalitozoon spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two M. myotis , two N. noctula and one V. murinus ). Positive samples were sequenced and showed homology with the genotypes Encephalitozoon cuniculi II and Encephalitozoon hellem 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of Encephalitozoon spp. detected in bats.

The aim of this study was to detect antibodies to Toxoplasma gondii and Neospora caninum in exotic animal species kept in three zoos in Slovakia. Antibodies to T. gondii and N. caninum were detected by commercial Enzyme-linked immunosorbent assay (ELISA, ID Screen Toxoplasmosis Indirect Multispecies and ID Screen Neospora caninum Indirect Multispecies, ID Vet, Montpellier, France). Antibodies to T. gondii and N. caninum were detected in 43% (24/56) and 5% (3/55) of animals, respectively. The three animals with N. caninum antibodies: two wolves (Canis lupus) and one Hartmann's mountain Zebra (Equus zebra hartmannae), were clinically healthy, and both wolves simultaneously had antibodies to T. gondii. The results of our study provide a picture of the recent circulation of T. gondii in three Slovakian zoos with the S/P (ratio of antibodies in the sample to antibodies in positive control) value higher than 200%, found in five animals (9%) indicating acute toxoplasmosis. The highest S/P value (296%) was detected in a Roloway monkey (Cercopithecus roloway), which was healthy without clinical signs, presuming that Roloway monkey is a species less susceptible to T. gondii infection. Results of our study showed the presence of T. gondii and N. caninum in Slovakian zoos, confirming recent T. gondii infections according to the high level of antibodies detected in five animals, referring to acute toxoplasmosis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)