First Results on the Presence of Mycotoxins in the Liver of Pregnant Fallow Deer (Dama dama) Hinds and Fetuses.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.

The impact of the striped field mouse's range expansion on communities of native small mammals.

Understanding species expansion as an element of the dispersal process is crucial to gaining a better comprehension of the functioning of the populations and the communities. Populations of the same species that are native in one area could be considered nonindigenous, naturalised or invasive somewhere else. The striped field mouse has been expanding its range in south-western Slovakia since 2010, although the origin of the spread has still not been clarified. In light of the striped field mouse's life history, the recent range expansion is considered to be the expansion of a native species. This study analyses the impact of the striped field mouse's expansion on the native population and small mammal communities and confronts the documented stages of striped field mouse expansion with the stages of invasion biology. Our research replicates the design and compares results from past research of small mammals prior to this expansion at the same three study areas with the same 20 study sites and control sites. Several years after expansion, the striped field mouse has a 100% frequency of occurrence in all study sites and has become the dominant species in two of the study areas. The native community is significantly affected by the striped field mouse's increasing dominance, specifically: (i) we found a re-ordering of the species rank, mainly in areas with higher dominance, and (ii) an initial positive impact on diversity and evenness during low dominance of the striped field mouse turned markedly negative after crossing the 25% dominance threshold. Results suggested that the variation in the striped field mouse's dominance is affected by the northern direction of its spread. Our findings show that establishment in a new area, spread and impact on the native community are stages possibly shared by both invasive and native species during their range expansion.

Effectiveness Regarding Hantavirus Detection in Rodent Tissue Samples and Urine.

The natural hosts of Orthohantaviruses are rodents, soricomorphs and bats, and it is well known that they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces throughout the entirety of their lives. We aim to identify the effectiveness of hantavirus detection in rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 deceased animals. All organs and urine were tested using nested reverse transcription polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) by Western blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV and Tula virus (TULV) were clearly identified. Amid the PCR-positive samples, the nucleic acid of the viruses was detected most effectively in the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only three out of 20 rodent urine samples were positive when tested using nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid in any of the tested organs.

Temporal Dynamics of Two Pathogenic Hantaviruses Among Rodents in Hungary.

Hantaviruses are worldwide pathogens, which often cause serious or even fatal diseases in humans. Hosts are predominantly in the form of rodents and soricomorphs; however, bats are also described as an important reservoir. In Hungary, representatives of two human pathogenic species of the genus Orthohantavirus are present: the Dobrava-Belgrade orthohantavirus and Puumala orthohantavirus. In Hungarian forests, the dominant rodent species are Apodemus flavicollis, Apodemus agrarius, Apodemus sylvaticus, and Myodes glareolus, all of which are natural reservoirs comprising different hantaviruses. The aim of the study was to survey the prevalence of hantaviruses among rodent populations and examine the potential relationship regarding population densities, years, sex, and seroprevalence. Rodents were trapped at 13 sampling plots in a forest reserve located in the Mecsek Mountain range, Hungary, from March to October between 2011 and 2014. Rodent serum samples were tested for IgG antibodies against Dobrava-Belgrade virus and Puumala virus by enzyme-linked immunosorbent assay (ELISA) using a recombinant nucleocapsid protein. During the 4-year sampling period, 2491 specimens were tested and 254 (10.2%) proved seropositive for orthohantaviruses. In 2011, the seroprevalence among Apodemus spp. and M. glareolus was 17.2% (114/661) and 3.9% (3/77), respectively, although this rate had reversed itself in 2014. Seropositivity was substantiated in 18.4% (12/65) of Myodes voles, while only 3.6% (13/359) of the tested Apodemus rodents were found to be IgG positive. Seroconversion was observed in 58 cases, while seroreversion was only detected in 3 individual cases. A significant difference among the number of infected males and females was identified in the first 2 years of our study. Winter survival with respect to rodents was not negatively affected due to the hantavirus infection. Hantavirus seroprevalence was not directly influenced by host abundance. Consequently, we assume that high rodent density alone does not lead to an increased risk of hantavirus infection among the rodent host population.

Serologic survey of the Crimean-Congo haemorrhagic fever virus infection among wild rodents in Hungary.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne pathogen, which causes an increasing number of severe infections in many parts of Africa, Asia and in Europe. The virus is primarily transmitted by ticks, however, the spectrum of natural hosts regarding CCHFV includes a wide variety of domestic and wild animals. Although the presence of CCHFV was hypothesized in Hungary, data in support of CCHFV prevalence has thus far, proven insufficient. In the present study, rodents belonging to four species, the yellow-necked mouse (Apodemus flavicollis), the striped field mouse (A. agrarius), the wood mouse (A. sylvaticus) and the bank vole (Myodes glareolus), were all systematically trapped in the Mecsek Mountain region (Southwest Hungary), from 2011 through 2013. Rodent sera were collected and screened for CCHFV antibodies with dot-blot pre-screening and immunofluorescence assay. Among the 2085 tested rodents, 20 (0.96%) were positive for IgG antibody against CCHFV. Seroprevalence was the highest (1.25%) in A. flavicollis serum samples. Distinctly, we now provide the first data regarding CCHFV occurrence and seroprevalence among wild rodents in Hungary. This observation represents a need for large-scale surveillance to effectively assess the enzootic background and the potential public health risk of CCHFV in Hungary.

Hepatitis E virus in Common voles (Microtus arvalis) from an urban environment, Hungary: Discovery of a Cricetidae-specific genotype of Orthohepevirus C.

Hepatitis E virus is a major causative agent of acute hepatitis worldwide. Despite its zoonotic potential, there is limited information about the natural chain of hepevirus infection in wildlife, and the potential reservoir species. In this study, we performed a HEV survey by heminested RT-PCR on rodent samples from an urban environment (in the city of Pécs, Hungary) and investigated the prevalence of the virus among these native rodent species (Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Microtus arvalis and Myodes glareolus). HEV was detected exclusively in Common voles (M. arvalis), in 10.2% of screened voles, and 3.2% of all investigated samples from all species. Based on the phylogenetic analysis, our strain showed the closest homology with European Orthohepevirus C strains detected previously in faecal samples of birds of prey and Red fox, supporting the possibility of the dietary origin of these strains. In addition, our samples showed close phylogenetic relation with a South American strain detected in Necromys lasiurus (Cricetidae), but separated clearly from other Muridae-associated strains, suggesting the presence of a Cricetidae-specific genotype in Europe and South-America. Based on these results, we hypothesize the reservoir role of M. arvalis rodents for the European Cricetidae-specific Orthohepevirus C genotype.

Parallel Survey of Two Widespread Renal Syndrome-Causing Zoonoses: Leptospira spp. and Hantavirus in Urban Environment, Hungary.

Rodents are important reservoir hosts for several zoonotic pathogens that cause significant morbidity and mortality in humans. Among others, leptospirosis is one of the most widespread zoonotic diseases worldwide and has the similar clinical manifestation with hantavirus infection in humans. Despite the fact that both pathogens have great epidemiological significance in Europe, no epizootiological data exist for urbanized areas so far. Therefore, this study was conducted to investigate the occurrence and prevalence of Leptospira spp. and hantaviruses in small wild rodents living in close proximity to humans. Altogether, 338 small rodents representing five different species (Apodemus agrarius, A. flavicollis, A. sylvaticus, Microtus arvalis, and Myodes glareolus) were captured in the city of Pécs (Hungary) and screened for pathogens by different types of PCR methods (TaqMan-based real-time PCR/PCR, RT-PCR/PCR). A total of 18.3% of the rodents were positive for Leptospira kirschneri, L. interrogans, and L. borgpetersenii. Nucleic acid of Tula hantavirus and human pathogen Dobrava-Belgrade orthohantavirus were detected in 8% of tested specimens. Furthermore, dual infections with both Leptospira spp. and hantaviruses were shown in 2.6% of animals, suggesting that the same rodent host can be infected with several pathogens at the same time, therefore, representing a serious threat to public health. Overall, this study provides important surveillance data on the prevalence of Leptospira spp. and hantaviruses from rodents in urbanized environment for the first time in Hungary and emphasizes the importance of further ecoepidemiological investigations.

Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis).

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1-L3, M2-M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

Serologic survey of orthopoxvirus infection among rodents in hungary.

As a result of discontinuing vaccination against smallpox after the late 1970s, different orthopoxviruses (OPVs), such as cowpox virus (CPXV), have become a re-emerging healthcare threat among zoonotic pathogens. In Hungary, data on OPV prevalence among its rodent host species have been absent. Here, rodents belonging to four species, i.e., striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), wood mouse (A. sylvaticus) and bank vole (Myodes glareolus), were live trapped at 13 sampling plots on a 149-ha area in the Mecsek Mountains, Hungary, from March to September in 2011 and 2012. Rodent sera were collected and screened for OPV-reactive antibodies with an immunfluorescence assay (IFA). Among the 1587 tested rodents, 286 (18.0%) harbored OPV-specific antibodies. Seroprevalence was the highest for the bank vole (71.4%) and the striped field mouse (66.7%). Due to a masting event in the autumn of 2011 across Central Europe, the abundance of bank voles increased drastically in the 2012 season, raising the overall OPV seroprevalence. We provide the first data on OPV occurrence and seroprevalence in rodents in Hungary. The circulation of OPV in rodents in densely populated areas warrants further studies to elucidate the zoonotic potential of OPV in humans.

Serosurvey of pathogenic hantaviruses among forestry workers in Hungary.

OBJECTIVES: The aim of the study was to survey the prevalence of human hantavirus infections among forestry workers, who are considered a risk population for contracting the disease. Sera collected from volunteers were tested for antibodies against Dobrava-Belgrade (DOBV) and Puumala (PUUV) viruses. MATERIAL AND METHODS: For serological analyses, full capsid proteins of DOBV and PUUV viruses were produced in a bacterial expression system, while Ni-resin was used for protein purification. Samples were screened for anti-hantavirus antibodies by ELISA, results were confirmed by Western blot analysis. RESULTS: A total of 835 samples collected from 750 males and 85 females were tested by indirect ELISA and positive test results were confirmed by Western blot assay. Out of the 45 ELISA-reactive samples, 38 were confirmed by Western blot analysis. The regional distribution of seropositive individuals was as follows: 1.9% (2/107) in the Danube-Tisza Plateau (Great Plains), 3.1% (10/321) in the Southern Transdanubian region, 5.2% (13/248) in the Northern Transdanubian, and 8.2% (13/159) in the North Hungarian Mountains. CONCLUSIONS: Our data show marked geographic differences in seroprevalence of pathogenic hantaviruses within Hungary, indicating elevated exposure to hantavirus infections in some areas.

Molecular detection and phylogenetic analysis of tick-borne encephalitis virus in rodents captured in the transdanubian region of Hungary.

Abstract Tick-borne encephalitis virus (TBEV) infection is a common zoonotic disease affecting humans in Europe and Asia. To determine whether TBEV is present in small mammalian hosts in Hungary, liver samples of wild rodents were tested for TBEV RNA. Over a period of 7 years, a total of 405 rodents were collected at five different geographic locations of the Transdanubian region. TBEV nucleic acid was identified in four rodent species: Apodemus agrarius, A. flavicollis, Microtus arvalis, and Myodes glareolus. Out of the 405 collected rodents, 17 small mammals (4.2%) were positive for TBEV. The present study provides molecular evidence and sequence data of TBEV from rodents in Hungary.

Molecular characterization of Dobrava and Kurkino genotypes of Dobrava-Belgrade hantavirus detected in Hungary and Northern Croatia.

Among the Hantavirus genus, Saaremaa virus (SAAV) has been the subject of taxonomical debates. While the International Committee on Taxonomy of Viruses declares SAAV as a distinct species, several European hantavirus experts proposed that SAAV is in fact a genotype of Dobrava-Belgrade virus (DOBV). In the present study we performed S-segment-based phylogenetic analysis of eight DOBV strains identified in rodents in Hungary and Northern Croatia. These new sequences considerably increase the number of complete nucleoprotein gene sequences deposited in the NCBI database. Our phylogenetic analysis clearly support the taxonomical nomenclature recently proposed for DOBV, i.e., genotypes such as Dobrava, Saaremaa, Kurkino, and Sochi should indeed be classified within the DOBV hantavirus species. Moreover, we found that only the Dobrava and Kurkino genotypes of DOBV species are circulating in Hungary while currently there is no evidence for the presence of Saaremaa genotype.

Does malaoxon play a role in the geno- and cytotoxic effects of malathion on human choriocarcinoma cells?

This investigation was undertaken to elucidate whether the active metabolite of malathion, malaoxon, has any role in exerting cyto- and genotoxic effects for human choriocarcinoma (JAR) cell line which is an acceptable model for human placental cells. Gas chromatography-mass spectrometry (GC-MS) analysis were separately performed on the cell compartment and supernatant cell culture medium after subjecting the cell line to different malathion concentrations (10-400 µg/mL) and for various incubation periods (0.5 to 24 hours). GC-MS analysis showed that the sonication performed for the disruption of the cells did not cause the chemical change of malathion. The uptake of malathion by the cells was relatively fast. However, the presence of malaoxon, even in trace amounts, could not be confirmed either in samples originating from disrupted cells or in the cell culture medium. Although the hydrolysis of malaoxon occurred in the culture medium, this degradation process could not be counted as a reason for the absence of malaoxon. Since both malathion and malaoxon standard compounds could be accurately detected and distinguished by the applied liquid-liquid extraction and GC-MS methods, one can conclude that, in the case of JAR cells, the parent compound, (i.e. malathion itself) is responsible for the observed in vitro cyto- and genotoxic effects. Our results indicate that the direct toxicity of malathion contributes to the complications of pregnancy observed for environmental malathion exposure.

Detection of Dobrava-Belgrade hantavirus using recombinant-nucleocapsid-based enzyme-linked immunosorbent assay and SYBR Green-based real-time reverse transcriptase-polymerase chain reaction.

Dobrava (DOBV) hantaviruses belong to the genus Hantavirus, family Bunyaviridae, and are carried by yellow-necked and striped field mice. The goal of this study was to detect DOBV using serological and genetic methods in Apodemus rodents in Hungary and in northern Croatia. During the study period, a total of 125 Apodemus sp. (67 A. agrarius, 58 A. flavicollis) were tested for the presence of hantaviruses, and 21 rodents (17%) were positive by rRT-PCR and/or ELISA. We conclude that the prevalence of DOBV is much higher than previously anticipated. The simultaneous use of molecular and serological techniques provides a highly reliable way to detect hantavirus infections.

Extramedullary hematopoiesis is dysregulated in histamine-free histidine decarboxylase knockout (HDC-/-) mice.

OBJECTIVE AND DESIGN: In this study we investigated the role of histamine on the extramedullary hematopoiesis. METHODS: Male histidine decarboxylase knockout (HDC(-/-)) mice and wild-type mice were used (n = 5/group). Groups of mice received sublethal total-body gamma irradiation at a single dose of 4 Gy. Spleen cells were studied at different time points post-irradiation by flow cytometry, colony forming unit (CFU) assay, and real-time PCR. For statistical analysis Student's t test, ANOVA, and Holm-Sidak post-hoc test were used. RESULTS: By day 14 after irradiation, spleen cell counts increased almost eightfold in wild-type and not even fourfold in HDC(-/-) mice (P < 0.01). The proliferative capacity and interleukin-3 signaling of stem cells were impaired in HDC(-/-) mice. STAT5 mRNA expression was decreased in granulocyte-myeloid colonies by 72.9 +/- 8.6% (P < 0.001), compared to the wild-type. CONCLUSIONS: The absence of histamine adversely affects splenic hematopoiesis via direct and indirect mechanisms.

First detection of Tula hantaviruses in Microtus arvalis voles in Hungary.

Tula hantavirus (TULV) is a member of the genus Hantavirus, family Bunyaviridae and is mainly carried by the European common vole (Microtus arvalis). In order to detect TULV, we tested Microtus arvalis (MAR) and Microtus subterraneus (MSU) voles captured in two different locations of the Southern Transdanubian region of Hungary. The viral genome was detectable in 37% of the tested MAR voles but, interestingly, was absent in all MSU. Phylogenetic analysis performed with a partial coding sequence of the capsid gene showed that Hungarian TULV strains clustered with viruses detected in western Slovakia and in the Czech Republic. To the best of our knowledge, this is the first report on the identification of TULV detected in MAR voles in the Transdanubian region of Hungary.

First detection of Dobrava hantavirus from a patient with severe haemorrhagic fever with renal syndrome by SYBR Green-based real time RT-PCR.

Dobrava hantavirus (DOBV) infection was diagnosed in a previously healthy 46-y-old hunter suffering from severe haemorrhagic fever with renal syndrome (HFRS). Specific IgM antibodies against DOBV were identified by an immunofluorescence assay, while viral nucleic acid was detected by the molecular method, confirming the diagnosis. Our results reveal an existing risk of DOBV transmission to humans in Hungary.

Detection of Dobrava hantaviruses in Apodemus agrarius mice in the Transdanubian region of Hungary.

Dobrava hantavirus (DOBV) belongs to the genus Hantavirus of the family Bunyaviridae, and is carried by yellow necked and striped field mice (Apodemus flavicollis and Apodemus agrarius), respectively. The aim of this study was to detect and genetically characterize new DOBV strains in rodents captured in the Transdanubian region of Hungary. Rodent corpses were dissected and lung tissues were used for hantavirus detection by SYBR Green-based real-time RT-PCR using specific primers located in the S-segment of the virus genome. A total of 22 captured animals of the Apodemus species were tested for the presence of DOBV. Three out of the 22 mice were positive. Phylogenetic and molecular sequence analyses showed that Hungarian DOBVs were most closely related to those viruses detected from A. agrarius mice in Slovenia. Based on our new data from the region we concluded that extended reservoir studies would be necessary in the future.

Histamine H1 and H2 receptors but not H4 receptors are upregulated during bone marrow regeneration.

The role of histamine receptors in radiation-induced bone marrow (BM) regeneration was investigated with aspects of functional genomics. H1R and H2R mRNA expression increased during regeneration in both histidine decarboxylase knockout (HDC-/-) and wild type (HDC+/+) mice, though to a lesser extent in HDC-/- mice. H4R mRNA expression was downregulated in both groups. Mainly CD34+ cells were responsible for the elevation of intracellular histamine and HDC content in HDC+/+ BM cell populations. The differential changes in the expression of its receptors, and also its elevated levels in hematopoietic progenitors support the regulatory role of histamine in BM regeneration, that could be further explored by future gene expression studies.