Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Detection of circovirus in free-ranging brown rats (Rattus norvegicus).

Accidentally found, two poisoned brown rats from Hungary were surveyed for presence of circoviral DNA, using specific nested primers, designed against the rep gene of the virus. Both specimens were positive. The whole genomes were amplified using inverse PCR based on the Rep sequence parts and sequenced by the primer walking method. Genomic analyses revealed that these novel rat viruses, together with tawny owl-associated circovirus reported by Italian researchers in 2022, are sequence variations of the same virus from genus Circovirus. In phylogenetic reconstructions, these circovirus strains detected from brown rats clustered closest to circoviruses derived from faeces samples of various predatory mammals. Molecular data as well as the phylogenetic analyses of the complete derived replication-associated protein and the capsid protein, as well as the prey preference of the host species of the recently described tawny owl-associated virus suggest that brown rat could be the evolutionary adapted host of the viruses described in this paper (brown rat circovirus types 1 and 2) and the previously reported tawny owl-associated virus. Possible pathogenic and zoonotic role of these viruses need further studies.

Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men.

OBJECTIVES: Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. METHODS: We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. RESULTS: In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. CONCLUSIONS: We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

Hydrogen-dependent denitrification in a two-reactor bio-electrochemical system.

An autotrophic biological process was developed for the treatment of nitrate-contaminated drinking water. The system comprised of two steps: the water to be treated was first enriched with hydrogen (energy source) in the cathodic chamber of an electrochemical cell, and then denitrified in the bioreactor. The bioreactor was a packed bed of granulated activated carbon, and the water flow was directed in an upward continuous mode. The system was operated for one year, at various water velocities and current intensities. Denitrification rates up to 0.25 kg N m-3 d-1 were obtained at the hydraulic residence time of 1 h. The system was stable. When detected in the effluent, the concentration of nitrite was low, even under conditions that resulted in the elution of very high concentrations of nitrate.

Milkborne campylobacter infection in Hungary.

In April 1998, an annual 2-day animal farm sale was held in Hódmezóvásárhely, where 500 to 600 visitors consumed unpasteurized milk. The first signs of disease began 2 days after the end of the sale. Fifty-two people from a wide age range fell ill, primarily with inflammatory enteritis. These cases included 34 with Campylobacter positivity: 30 with Campylobacter jejuni and 4 with Campylobacter coli. Environmental samples (raw milk, udder swabs, and rectal swabs from 12 cows in the suspected herd) were tested 2 weeks after the first signs of the disease, and two rectal swabs were found to be positive for C. jejuni. Initially, the epidemic seemed to be sporadic and, accordingly, only 26 human and 2 animal Campylobacter isolates were reserved for randomly amplified polymorphic DNA analysis. This comparative analysis verified that fecally contaminated milk was the source of the outbreak. The DNA-banding patterns of 20 C. jejuni isolates (19 human and 1 animal) were identical. The antibiotic susceptibilities of the Campylobacter isolates were determined, and only six C. jejuni (human) isolates, one C. coli (human) isolate, and one C. jejuni (animal) isolate were resistant to tetracycline, both by disk diffusion and by E test (antimicrobial gradient strip for the quantitative determination of susceptibility or resistance of microorganisms). No plasmid was detected in these tetracycline-resistant isolates. The endotoxin production of Campylobacter isolates was examined via the cytopathogenic effect on the Vero cell line. This effect exhibited various degrees of positivity in 19 cases. Only two human C. jejuni isolates displayed + + + + positivity. Both isolates were from patients who had required antibiotic therapy and hospital care.

Directional cloning of native PCR products with preformed sticky ends (autosticky PCR).

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5' ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5' single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This "autosticky PCR" (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5' overhang.

Correlations between the tissue redox-state and K(+)-contractures.

Analyzing the mechanisms of redox-modulation of the excitatory-contractory process, recently the amplitude of K(+)-contractures, tissue redox-state potential and electrical burst activity were simultaneously measured in the rectus abdominis muscle of the frog (Rana esculenta) following oxidant (thionine) and reductant (ascorbate) treatments. Pretreatment with oxidant in parallel with the increment of redox-state potential increased, while pretreatment with reductant, parallel with the decrement of redox-state potential decreased significantly both the amplitudes of K(+)-contractures and the electrical burst activity. The main mechanisms of action of this phenomenon, at least of the phasic portion, in all probability is the increase of intracellular quotient of the ionized/bound calcium after oxidizing, but a decrease of this quotient following reducing shifts. In the case of tonic portion an increase of Ca2(+)-influx through the Na(+)-Ca2(+)-exchange diffusion mechanisms seems feasible. Other mechanisms are also discussed. Hence, the mechanism of K(+)-contractures is under the control of tissue redox-state potential as well.