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We aimed to estimate the proportion of the population infected with SARS-CoV-2 in the first year of the pandemic. The study population consisted of outpatient adults with mild or no COVID-19 symptoms, and was divided into subpopulations with different levels of exposures. Of the subpopulation without known previous COVID-19 contacts 4143, of the subpopulation with known COVID-19 contacts 594 persons were investigated. IgG- and IgA-seroprevalence and RT-PCR positivity were determined in context with COVID-19 symptoms. We hope to have contributed to the understanding of the significance of the asymptomatic and mild infections in the long persistence of the pandemic.

In this research uterine swab and biopsy samples were collected from 40 infertile dairy cows kept at five dairy cattle farms in Hungary. Samples were tested for bacteria including Coxiella burnetii chlamydiae, Mycoplasma and Ureaplasma, and for the viruses Bovine herpesvirus 1 (BoHV-1) and Bovine viral diarrhoea virus (BVDV). Chlamydiaceae DNA was detected by real-time PCR in 22/40 (55%) samples. Coxiella burnetii DNA was detected in 3/40 (7.5%) cases by real-time PCR. Mycoplasma and Ureaplasma DNA was found in 2/40 (5%) and 4/40 (10%) cows, respectively. BVD and BoHV-1 DNA was not detected in any samples. Escherichia coli as a recognised uterine pathogen was found in two cases. The following potential uterine pathogens were found: Bacillus licheniformis (one case), non- haemolytic streptococci (five cases), Histophilus somni (two cases) and Candida krusei (two cases). Blood samples were collected at same time as swab samples from all 40 cows, and their examination for C. burnetii antibodies by ELISA revealed seropositivity in 26/40 cows (65%). Histological examination of the uterine biopsy samples showed the presence of mild lympho-histiocytic infiltration in the mucosain 22 cases (59%). Moderatelympho-histiocytic infiltration of the endometrium was evident in 13 cases (35%), while in two cases (6%) severe inflammatory cell infiltration of the endometrium with lympho-histiocytes and neutrophil granulocytes was found. Although no statistical correlation could be demonstrated between the severity of histological lesions of the endometrium and the uterine pathogenicity of the bacteria (P = 0.8555), endometritis of a certain severity grade and/or a recognised or potential uterine pathogen were found in all samples. The latter may play a role in the development of infertility either collectively or independently., U ovom su istraživanju prikupljeni brisevi maternice i biopsijski uzorci 40 neplodnih mliječnih krava s pet mliječnih farmi u Mađarskoj. Uzorci su testirani na bakterije, uključujući; Coxiella burnetii, klamidiju, mikoplazmu i ureaplazmu te na viruse uključujući goveđi herpesvirus 1 (BoHV- 1) i virus virsnog proljeva goveda (BVDV). DNK Chlamydiaceae otkriven je PCR testom u stvarnom vremenu u 22/40 (55 %) uzoraka. DNK bakterije Coxiella burnetii otkriven je u 3/40 (7,5 %) slučajeva PCR testom u stvarnom vremenu. DNK mikoplazme i ureaplazme pronađen je u 2/40 (5 %), odnosno 4/40 (10 %) krava. DNK virusa BVD i BoHV-1 niti u jednom uzorku nije otkriven. Escherichia coli kao priznati maternični patogen pronađen je u dva slučaja. Pronađeni su sljedeći potencijalni maternični patogeni: Bacillus licheniformis (jedan slučaj), nehemolitički streptokoki (pet slučajeva), Histophilus somni (dva slučaja) i Candida krusei (dva slučaja). Uzorci krvi su istovremeno prikupljeni kad i brisevi od svih 40 pokusnih krava. Njihova pretraga na protututijela C. burnetii ELISA metodom otkrila je seropozitivnost u 26/40 krava (65 %). Histološka pretraga uzoraka biopsije maternice pokazala je prisutnost blage limfohistiocitotske infiltracije u sluznici u 22 slučaja (59 %). Umjerena limfohistiocitotska infiltracija endometrija bila je prisutna u 13 slučajeva (35 %), dok je u dva slučaja (6 %) otkrivena ozbiljna upalna stanična infiltracija endometrija s limfohistiocitima i neutrofilnim granulocitima. Premda nije bilo moguće dokazati statističku korelaciju između ozbiljnosti histoloških lezija endometrija i maternične patogenosti bakterija (P = 0,8555), endometrioza određenog stupnja ozbiljnost i/ ili priznati ili potencijalni maternični patogeni pronađeni su u svim uzorcima. Ovi posljednji mogu prouzročiti razvooj neplodnosti bilo skupno ili neovisno.

Objective In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Results In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the “regulation of signaling receptor activity” Gene Ontology annotation as a result of viral infection.

Q fever is one of the commonest infectious diseases worldwide. A Coxiella burnetii prevalence of 97.6% has been found by ELISA and PCR tests of the bulk tank milk in dairy cattle farms of Hungary. The herd- and individual-level seroprevalence rates of C. burnetii in the examined dairy cows and farms have dramatically increased over the past ten years. Three high-producing industrial dairy farms were studied which had previously been found ELISA and PCR positive for C. burnetii by bulk tank milk testing. Coxiella burnetii was detected in 52% of the 321 cows tested by ELISA. Pregnancy loss was detected in 18% of the cows between days 29–35 and days 60–70 of gestation. The study found a higher seropositivity rate (80.5%) in the cows that had lost their pregnancy and a seropositivity of 94.4% in the first-bred cows that had lost their pregnancy at an early stage. The ELISA-positive pregnant and aborted cows were further investigated by the complement fixation test (CFT). In dairy herds an average of 66.6% individual seropositivity was detected by the CFT (Phase II) in previously ELISA-positive animals that had lost their pregnancy and 64.5% in the pregnant animals. A higher (Phase I) seropositivity rate (50.0%) was found in the cows with pregnancy loss than in the pregnant animals (38.5%). The high prevalence of C. burnetii in dairy farms is a major risk factor related to pregnancy loss.

Background Recent studies have disclosed the genome, transcriptome, and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for noncanonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (multiplicity of infection [MOI] = 0.1) applied for the infection. It has never been tested whether the alteration in the host gene expressions is caused by aging of the cells or by the viral infection. Findings In this study, we used Oxford Nanopore's direct cDNA and direct RNA sequencing methods for the generation of a high-coverage, high temporal resolution transcriptomic dataset of SARS-CoV-2 and of the primate host cells, using a high infection titer (MOI = 5). Sixteen sampling time points ranging from 1 to 96 hours with a varying time resolution and 3 biological replicates were used in the experiment. In addition, for each infected sample, corresponding noninfected samples were employed. The raw reads were mapped to the viral and to the host reference genomes, resulting in 49,661,499 mapped reads (54,62 Gbs). The genome of the viral isolate was also sequenced and phylogenetically classified. Conclusions This dataset can serve as a valuable resource for profiling the SARS-CoV-2 transcriptome dynamics, the virus–host interactions, and the RNA base modifications. Comparison of expression profiles of the host gene in the virally infected and in noninfected cells at different time points allows making a distinction between the effect of the aging of cells in culture and the viral infection. These data can provide useful information for potential novel gene annotations and can also be used for studying the currently available bioinformatics pipelines.

Recent studies have disclosed the genome, transcriptome and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for non-canonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (MOI=0.1) applied for the infection. In this study, we used direct cDNA and direct RNA nanopore sequencings for the generation of high-coverage, high-temporal-resolution transcriptomic datasets on SARS-CoV-2 and on primate host cells infected with a high virus titer (MOI=5). Sixteen sampling time points ranging from 1 to 96h with a varying time resolution and three biological replicates were used in the experiment for both the infected and the non-infected cells.

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

The goal of this study was to evaluate the suitability of a commercially available D-dimer assay as a diagnostic tool for testing dogs. This assay is an immunoturbidimetric diagnostic test, capable of determining the D-dimer levels in human plasma by using 2B9 monoclonal antibody. Plasma samples of clinically healthy (n = 20) and tumour-bearing (n = 50) dogs were measured. The tumours were grouped on the basis of histological type and aggressiveness, and then the measured D-dimer concentrations of these groups were compared to those of the control group. The differences were analysed statistically. For benign tumours, we did not find alterations in the D-dimer levels. However, in the case of malignant tumours (lymphoma, sarcoma, and carcinoma) and in the presence of metastases, significantly elevated D-dimer levels were measured. The assay proved to be suitable for measuring the D-dimer levels in plasma samples of dogs. The calculated reference range for dogs was confirmed to be between 0.06 and 0.69 µg/mL fibrinogen equivalent unit.

Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the vaccinia virus (VACV) transcriptome. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighbouring genes. This study provides new insights into the transcriptomic profile of this viral pathogen.

A short alternative open reading frame named ORF7a has recently been discovered within the nucleocapsid gene of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. Proteins (7ap) translated from the ORF7a of two divergent strains — a type I and a type II — are able to completely reduce the motility of nucleic acids at relatively high molar charge ratios in gel retardation assays indicating strong dsDNA- and ssRNA-binding capability. Conserved RNA- and DNA-binding properties suggest that nucleic acid binding is a functional property of the divergent 7aps, and not an arbitrary consequence of their net positive charge. Sera from Hu7ap-immunised pigs and mice did not react with Hu7ap or Hu7ap-GFP; however, antinuclear antibodies were detected in the sera of the immunised animals, suggesting an ability of Hu7ap to interact with or mimic autoantigenic macromolecules.

Characterization of global transcriptomes using conventional short-read sequencing is challenging because of the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps, etc. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the dynamic vaccinia virus (VACV) transcriptome and assess the effect of viral infection on host gene expression. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighboring genes. We distinguished five classes of host genes according to their temporal responses to viral infection. This study provides novel insights into the transcriptomic profile of a viral pathogen and the effect of the virus on host gene expression.Author SummaryViral transcriptomes that are determined using conventional (first- and second-generation) sequencing techniques are incomplete because these platforms are inefficient or fail to distinguish between types of transcripts and transcript isoforms. In particular, conventional sequencing techniques fail to distinguish between parallel overlapping transcripts, including alternative polycistronic transcripts, transcriptional start site (TSS) and transcriptional end site (TES) isoforms, and splice variants and RNA molecules that are produced by transcriptional read-throughs. Long-read sequencing (LRS) can provide complete sets of RNA molecules, and can therefore be used to assemble complete transcriptome atlases of organisms. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome contains large numbers of TSSs and TESs for individual viral genes and has a high degree of polycistronism, together leading to enormous complexity. In this study, we applied single molecule real-time and nanopore-based cDNA and direct-RNA sequencing methods to investigate transcripts of VACV and the host organism.

Several trematodes that parasitize vertebrate animals utilize swimming aquatic larvae to infect the host percutaneously. The most important ones among these parasites are the blood-flukes of birds and mammals comprising species that are also zoonotic. Within this latter group are species that cause the bilharziasis or schistosomiasis of inhabitants of the tropical countries, and other trematode species that are able to penetrate human skin, but do not develop to an adult form of the worm in the body. In temperate climates this latter type of infection occurs mainly in the form of an unpleasant inflammation of the skin and is often called “swimmer’s itch”. In most of these cases, the origin of the larvae remains unexplored, the source of the infection is neglected by the medical or veterinarian practitioners. Herein we report for the first time in Hungary that the cause of such dermatitis was the cercariae of Schistosoma turkestanicum, which infected red deer (Cervus elaphus) in this country. The local name of this pristine disease is “water mange” and it occurs only in one of the floodplains of the Danube. On the basis of informal communication this symptom seems to be rather regular among people who do fishing or have a bath in the habitat of the blood-fluke. In the case of adequate anamnesis it is worth examining the origin of the cercarial dermatitis which may give cross-reactions with human schistosomiasis during serological tests. Orv. Hetil., 2016, 157(40), 1579–1586.

Background Poxviruses are large DNA viruses that infect humans and animals. Vaccinia virus (VACV) has been applied as a live vaccine for immunization against smallpox, which was eradicated by 1980 as a result of worldwide vaccination. VACV is the prototype of poxviruses in the investigation of the molecular pathogenesis of the virus. Short-read sequencing methods have revolutionized transcriptomics; however, they are not efficient in distinguishing between the RNA isoforms and transcript overlaps. Long-read sequencing (LRS) is much better suited to solve these problems and also allow direct RNA sequencing. Despite the scientific relevance of VACV, no LRS data have been generated for the viral transcriptome to date. Findings For the deep characterization of the VACV RNA profile, various LRS platforms and library preparation approaches were applied. The raw reads were mapped to the VACV reference genome and also to the host (Chlorocebus sabaeus) genome. In this study, we applied the Pacific Biosciences RSII and Sequel platforms, which altogether resulted in 937,531 mapped reads of inserts (1.42 Gb), while we obtained 2,160,348 aligned reads (1.75 Gb) from the different library preparation methods using the MinION device from Oxford Nanopore Technologies. Conclusions By applying cutting-edge technologies, we were able to generate a large dataset that can serve as a valuable resource for the investigation of the dynamic VACV transcriptome, the virus-host interactions, and RNA base modifications. These data can provide useful information for novel gene annotations in the VACV genome. Our dataset can also be used to analyze the currently available LRS platforms, library preparation methods, and bioinformatics pipelines.

Three new serotypes were found among Rhodococcus equi strains, which could not be assigned into any of the seven serotypes of Prescott’s system. Fortythree R. equi strains out of 44 previously nontypable ones isolated in Hungary could be allocated into one of the three new serotypes using the agar gel immunodiffusion (AGID) test. The three new suggested serotypes are serotype 8 (proposed reference strain: HNCMB-138003), serotype 9 (proposed reference strain: HNCMB-138004) and serotype 10 (proposed reference strain: HNCMB-138005). Hyperimmune sera produced in rabbits against the new serotypes and reference strains gave precipitation only with their homologous antigens, and no crossreactions were observed. All of the previously nontypable isolates from clinical samples of horses (lung abscesses, intestinal lymph nodes, mediastinal lymph nodes) proved to be serotype 8, while strains of serotypes 8, 9 and 10 could be isolated from nasal and rectal swabs of horses and from the soil. Serotype 9 dominated among the previously nontypable strains of swine origin. One of the previously nontypable human strains was serotype 10. This serotype was also isolated from pigs, horses and the soil. The description of the three new serotypes can help us reveal new correlations between the host species, geographical origin and serotype of R. equi isolates.

The vaccinia virus is a large, complex virus belonging to the Poxviridae family. Here, we report the complete, annotated genome sequence of the neurovirulent Western Reserve laboratory strain of this virus, which was sequenced on the Pacific Biosciences RS II and Oxford Nanopore MinION platforms.

Purpose: Vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. We investigated whether arming vaccinia virus with interleukin-10 (IL10) could enhance the antitumor efficacy with the view that IL10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus. Experimental Design: The antitumor efficacy of IL10-armed vaccinia virus (VVLΔTK-IL10) and control VVΔTK was assessed in pancreatic cancer cell lines, mice bearing subcutaneous pancreatic cancer tumors and a pancreatic cancer transgenic mouse model. Viral persistence within the tumors was examined and immune depletion experiments as well as immunophenotyping of splenocytes were carried out to dissect the functional mechanisms associated with the viral efficacy. Results: Compared with unarmed VVLΔTK, VVLΔTK-IL10 had a similar level of cytotoxicity and replication in vitro in murine pancreatic cancer cell lines, but rendered a superior antitumor efficacy in the subcutaneous pancreatic cancer model and a K-ras-p53 mutant-transgenic pancreatic cancer model after systemic delivery, with induction of long-term antitumor immunity. The antitumor efficacy of VVLΔTK-IL10 was dependent on CD4+ and CD8+, but not NK cells. Clearance of VVLΔTK-IL10 was reduced at early time points compared with the control virus. Treatment with VVLΔTK-IL10 resulted in a reduction in virus-specific, but not tumor-specific CD8+ cells compared with VVLΔTK. Conclusions: These results suggest that VVLΔTK-IL10 has strong potential as an antitumor therapeutic for pancreatic cancer. Clin Cancer Res; 21(2); 405–16. ©2014 AACR.

Based on initial reports of infection with Francisella tularensis in dogs in northern Europe, a serological study in hunting dogs was conducted in Austria. Five (6.25%) tested positive. The results highlight the potential for disease transmission to dogs and raise the question of whether dogs could be a source of infection for humans.

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein ( omp) 31 gene–based part of the “Bruce-ladder” multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS) 711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS 711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS 711 could be an active transposon in this Brucella species.

Several trematodes that parasitize vertebrate animals utilize swimming aquatic larvae to infect the host percutaneously. The most important ones among these parasites are the blood-flukes of birds and mammals comprising species that are also zoonotic. Within this latter group are species that cause the bilharziasis or schistosomiasis of inhabitants of the tropical countries, and other trematode species that are able to penetrate human skin, but do not develop to an adult form of the worm in the body. In temperate climates this latter type of infection occurs mainly in the form of an unpleasant inflammation of the skin and is often called "swimmer's itch". In most of these cases, the origin of the larvae remains unexplored, the source of the infection is neglected by the medical or veterinarian practitioners. Herein we report for the first time in Hungary that the cause of such dermatitis was the cercariae of Schistosoma turkestanicum, which infected red deer (Cervus elaphus) in this country. The local name of this pristine disease is "water mange" and it occurs only in one of the floodplains of the Danube. On the basis of informal communication this symptom seems to be rather regular among people who do fishing or have a bath in the habitat of the blood-fluke. In the case of adequate anamnesis it is worth examining the origin of the cercarial dermatitis which may give cross-reactions with human schistosomiasis during serological tests. Orv. Hetil., 2016, 157(40), 1579-1586.

Sequence analysis revealed a short alternative open reading frame (ORF) named ORF7a within the nucleocapsid gene of genetically divergent porcine reproductive and respiratory syndrome virus (PRRSV) genomes. Alignment of the corresponding protein sequences (named 7ap) revealed substantial heterogeneity among 7aps of different genotypes, though all of them are predicted to be positively charged. Green fluorescent protein and FLAG fusion constructs of ORF7a of the HU-14432/2011 PRRSV demonstrated that 7ap is expressed. 7ap of HU- 14432/2011 (Hu7ap) was synthesised chemically, and ELISA experiments revealed that Hu7ap binds strongly to mammalian IgGs. Protein-protein gel retardation assays and complement fixation inhibition suggest that 7aps bind to the CH2 domain of the IgG(Fc) fragment. Cellular localisation and immunological characteristics of PRRSV 7ap may indicate multiple functions including nuclear and cytoplasmic over-tuning of normal cellular processes and immunosuppression.

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.

Francisella tularensis is a highly infectious zoonotic agent causing the disease tularemia. The common hamster (Cricetus cricetus) is considered a pest in eastern Europe, and believed to be a source of human tularemia infections. We examined the role of the common hamster in the natural cycle of tularemia using serologic methods on 900 hamsters and real-time polymerase chain reaction (PCR) on 100 hamsters in an endemic agricultural area. We collected 374 Ixodes acuminatus ticks from the hamsters and tested them by real-time PCR. All tests were negative. To examine clinical signs, pathology, and histopathology of acute tularemia infection similar to the natural infection, two hamsters were infected with a large dose of a wild strain of F. tularensis ssp. holarctica. After a short period of apathy, the animals died on the eighth and ninth days postinfection. The pathologic, histopathologic, and immunohistochemical examination contributed to the diagnosis of septicemia in both cases. Our results confirmed previous findings that common hamsters are highly sensitive to F. tularensis. We conclude that although septicemic hamsters may pose substantial risk to humans during tularemia outbreaks, hamsters in interepizootic periods do not act as a main reservoir of F. tularensis.

Summary Thirteen Francisella tularensis strains were isolated from 22 seropositive brown hares (Lepus europaeus) originating from different parts of Hungary, and further two from a patas monkey (Erythrocebus patas) and vervet monkey (Chlorocebus aethiops). The isolates were identified as F. tularensis ssp. holarctica on the basis of culture, morphological and biochemical characteristics. The identification was verified by polymerase chain reaction and the sequencing of the partial 16S rRNA gene. Utilization of carbon sources of the 15 F. tularensis strains was characterized with the Biolog system. The system was able to identify the strains already after 4 h of incubation, not only after the standard 24 h. After the analysis and comparison of the metabolic profiles of our strains with the Biolog database, it was concluded that not all carbon sources indicated in the database were utilized by our isolates. The Biolog software fails to distinguish the highly virulent F. tularensis ssp. tularensis and the moderately virulent F. tularensis ssp. holarctica but the Biolog microplates can be manually read to differentiate the two subspecies based on glycerol source utilization. As all the studied strains were unable to use glycerol, they could be identified as F. tularensis ssp. holarctica. The dendrogram based on the metabolic relationship of the strains shows that the isolates are very similar to each other, which correlates with the conservative genetic character of F. tularensis ssp. holarctica.

Attenuated vaccinia viruses (VV) selectively replicate in malignant cells and confer oncolytic effect in vivo. Here we demonstrate that oncolytic VV may also be used as a diagnostic agent for tumor-bearing mice. A series of recombinant vaccinia viruses has been constructed expressing optical reporters to mediate emission of bioluminescent and fluorescent light which can be visualized. Data show that following systemic virus delivery the developing tumors can be non-invasively visualized in mice in vivo. Renilla luciferase and Aquoria GFP have been effective in imaging xenografted PC-3 prostate and orthotopic MB-49 bladder tumors. Brighter reporters, Gaussia luciferase and Renilla GFP have been used for imaging TRAMP prostate cancer and C6 subcutaneous model of glioma. The C6 imaging data have been corroborated by traditional MRI. We are also developing a VV-mediated system for tumor detection in far red or near infra red fluorescent light. Results suggest that VV-mediated imaging is a promising alternative for early diagnosis of various human cancers.

Avipoxviruses are emerging pathogens affecting over 200 bird species worldwide. Genetic characterization of avipoxviruses is performed by analysis of genomic regions encoding the 4b and DNA polymerase. Whole genome sequence data are limited to a few avipoxvirus isolates. Based on phylogenetic analysis three major genetic clades are distinguished. In this study we report a novel avipoxvirus strain causing skin lesions in domestic turkey. The virus was identified in Hungary during 2011 in a flock of turkey vaccinated against avipoxvirus infection. The genome of the isolated strain, TKPV-HU1124/2011, was uniquely short (∼188.5kbp) and was predicted to encode reduced number of proteins. Phylogenetic analysis of the genes encoding the 4b and DNA polymerase separated TKPV-HU1124/2011 from other turkey origin avipoxviruses and classified it into a new genetic clade. This study permits new insight into the genetic and genomic heterogeneity of avipoxviruses and pinpoints the importance of strain diversity in vaccine efficacy.

Rhodococcus equi was isolated from lung, liver, spleen, and stomach content of two aborted equine fetuses of 7 and 8 months gestation from two different farms. Lesions included diffuse pyogranulomatous pneumonia with numerous Gram-positive coccobacilli within the cytoplasm of macrophages, multinucleated Langhans giant cells and neutrophils, and enhanced extramedullary hematopoiesis with megakaryocytosis within the liver and spleen. Detection of R. equi was made by bacteriology and immunohistochemistry for R. equi and VapA, the virulence factor of R. equi. R. equi and VapA were identified within the lungs of both fetuses, and its distribution correlated with lesions. Fetal lesions were similar to those observed in foals. We speculate that the fetuses contracted infection from the placenta by normal breathing movements or by swallowing of the amniotic fluid contaminated with R. equi.

Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-gamma receptor homolog (viroceptor).To facilitate the monitoring of virus properties, first we constructed a Lister vaccine strain derivative VV-RG expressing optical reporters. Further, we constructed a VV-RG derivative, VV-RG8, which lacks the IFN-gammaR viroceptor (B8R gene product). Replication, immunological and pathogenic properties of the constructed strains were compared.Viruses did not show significant differences in humoral and cellular immune responses of immune-competent mice. Replication of constructed viruses was efficient both in vitro and in vivo, but showed marked difference in kinetics of propagation. In cultured CV-1 epithelial cells, the VV-RG8 strain retained the propagation potential of the parental virus, while, in the C6 glial cells, significant delay was observed in the kinetics of the VV-RG8 replication cycle compared to VV-RG. The pathogenesis of the viruses was tested by survival assay and biodistribution in nude mice. High dose inoculation of nude mice with VV-RG8 caused less pronounced virus dissemination, improved weight gain, and increased survival rate, as compared with the VV-RG strain.The replication-competent virus VV-RG8 carrying a mutation at the B8R gene is less pathogenic for mice than the parental vaccine virus. We anticipate that step-wise inactivation of VV vaccine genes involved in evasion of host immune response may provide an alternative approach for generation of hyper-attenuated replication-competent vaccines.

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.

Purpose: We determined if vaccinia virus (VV) mediated delivery of human tumor suppressor p53 is safe and effective for bladder tumor therapy in an orthotopic murine model. Materials and Methods: We used recombinant VV (rVV) vectors to express transgenes in murine bladder cancer MB-49 cells in culture and those growing orthotopically in syngeneic mice. Cultured MB-49 cells were infected with rVV expressing reporter genes (rVV-L15) or p53 (rVV-TK-53) to measure virus infection and apoptosis induction. Orthotopic MB-49 tumors in C57/Bl6 mice were treated with intravesical instillation of rVV, and the tumor incidence, survival and transgene expression were determined. Results: Productive virus infection in vitro was observed in MB-49 cells, although at somewhat lower efficiency than in African Green Monkey kidney CV-1 cells (American Type Culture Collection, Manassas, Virginia). Expression of transgenes in vitro correlated with the virus dose. Cells infected with rVV underwent apoptosis with rVV-TK-53 inducing far greater cell death than rVV-L15. The rVV-L15 virus had no effect on tumor incidence but it increased mean survival compared with control. Instillation of rVV-TK-53 decreased the tumor incidence and 33% of mice survived treatment. At necropsy all nonsurviving mice had bladder tumor, whereas 2 survivors in the rVV-TK-53 treated group were tumor-free. Immunohistochemistry of tumors detected expression of the human p53 gene product in tumor cells. Conclusions: To our knowledge we report for the first time that recombinant vaccinia virus expressing human p53 can induce the death of MB-49 tumor cells in vivo, not only through the lytic effect of the virus, but also through expression of the death inducing p53 transgene. Further studies are needed to shed light on the mechanisms of rVV-TK-53 mediated tumor apoptosis and the antitumor immune response.

To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunization the spleen of the immunized mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium. Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA). The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland). Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG(1) subclass according to the heavy chain. Ten cell lines produced kappa and two produced lambda light-chain antibody. Possible cross-reactions of the produced monoclonal anti-M. bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure. All of the 12 antibodies tested gave a reaction with the antigen of M. bovis strain designated 26034. MAbs 3G12 (67, 69, and 72 kDa) and 5B8 (22, 25, and 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species. The other antibodies reacted with the M. bovigenitalium F7, M. anseris 8389, M. oculi, and M. gallisepticum s6 Holland antigens. Owing to its high specificity and affinity, the antibody produced by the cell line 3G12 is primarily considered suitable for use in immunodiagnostic tests of M. bovis infections.

Background Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease. Findings Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents. Conclusions Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

The present study describes the laboratory investigations after a single right eye drop (3.1 × 109 CFU live bacteria) immunisation of 6 dromedary camels (Camelus dromedarius) with live attenuated B. melitensis Rev 1 vaccine. The experiment was conducted over a period of 5 months. The vaccine strain was isolated for 16 days from only the right eye of the vaccinated dromedaries, but not from the left eye and both nostrils. Similar pattern of results was obtained by polymerase chain reaction. It was negative for the left eye, both nostrils (except for one dromedary) and for EDTA blood and serum. All vaccinated dromedaries seroconverted from day 16 after vaccination until 4 months shown by Rose-Bengal test and slide-agglutination test. No serological reactions were found after 5 months. The complement fixation test remained negative throughout the experiment. Information about the vaccination against brucellosis in camels, the within host disperse of the vaccine strain and the serological response are scarce. The experiment provided basic data about the feasibility of Brucevac conjunctival vaccine in camels. However, to prove if the immunised dromedaries acquired a lifelong immunity against brucellosis, pregnant vaccinated dromedaries need to be challenged with a field B. melitensis strain. We also recommend changing the conjunctival vaccination route to subcutaneous or intramuscular to prevent accidental infection due to B. melitensis vaccine strain excreted by lacrimation.

Blood samples were collected from 100 shepherd dogs, 12 hunting dogs and 14 stray dogs (apparently healthy) in southern Hungary to screen for the presence of emerging tick-borne pathogens. Based on real-time PCR results, 14 dogs (11%) had single or dual haemoplasma infection, and a same number of samples were positive for Anaplasma phagocytophilum. In one sample Coxiella burnetii was molecularly identified, and 20.3% of dogs seroconverted to the Q fever agent. Rickettsaemia (sensu stricto) was also detected in one animal. This is the first molecular evidence of autochthonous infection of dogs with the above pathogens in Hungary. The relatively high prevalence of haemoplasma and anaplasma infection among non-pet dogs is suggestive of a prolonged carrier status and bacteraemia of these animals rendering them epidemiologically significant as potential reservoirs and sentinels for tick-borne infections., Sándor Hornok was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext170. The study was sponsored by the TÁMOP-4.2.2.B-10/1 and TÁMOP-4.2.1.B-11/2/KMR-2011-0003 projects. Lilla Fekete, Miklós Gyuranecz and the study in part was supported by the Lendület programme (LP2012-22) of the Hungarian Academy of Sciences.

Data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete Freund’s adjuvant (CFA) that can maintain safe and durable immunologic homeostasis in NOD mice. Systemic coinoculation of prediabetic mice with recombinant vaccinia virus rVV-CTB::GAD and undiluted or 10-fold diluted CFA demonstrated a significant decrease in hyperglycemia and pancreatic islet inflammation in comparison with control animals during 17–61 and 17–105 weeks of age, respectively. Synergy in these beneficial effects was observed during 43–61 and 61–105 wks of age, respectively. Inflammatory cytokine and chemokine levels in GAD-stimulated splenocytes isolated from vaccinated mice were generally lower than those detected in unvaccinated mice. The overall health and humoral immune responses of the vaccinated animals remained normal throughout the duration of the experiments.

Q fever is an important zoonotic disease caused by Coxiella burnetii. There are few reliable data about C. burnetii infection available. The aim of this study was to assess the importance and potential infectious sources of Q fever in Hungary. A total of 215 milk samples (10 individual samples from each herd and 1 bulk tank milk sample from each cattle herd), and 400 serum samples (20 from each herd) were tested from 15 dairy cattle herds and 5 sheep flocks located in different parts of Hungary. The study found 19.3% (58/300) and 38.0% (57/150) seropositivity in cattle, and 0% (0/100) and 6.0% (3/50) seropositivity in sheep, by complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), respectively. C. burnetii DNA was detected by IS1111 element-based TaqMan real-time polymerase chain reaction (PCR) in 8.7% (13/150) of individual dairy cow milk samples, 4.0% (2/50) of individual sheep milk samples, and 66.7% (10/15) of dairy bulk tank milk samples. Samples taken from nine different commercially-available pasteurized cow milk products from different Hungarian producers were also tested for the presence of C. burnetii DNA, and eight of these samples were found to be positive (88.9%). The real-time PCR examination of 5402 ixodid ticks collected from different parts of the country yielded negative results. Knowledge of the true prevalence of Q fever is crucial for policymakers involved in evidence-based decision making.

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.