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The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kgThe antiarrhythmic effects of acute iv. (10 mg·kgDEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IChronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

Activation of the parasympathetic nervous system has been reported to have an antiarrhythmic role during ischemia–reperfusion injury by decreasing the arrhythmia triggers. Furthermore, it was reported that the parasympathetic neurotransmitter acetylcholine is able to modulate the ATP-dependent potassium current (I K-ATP), a crucial current activated during hypoxia. However, the possible significance of this current modulation in the antiarrhythmic mechanism is not fully clarified. Action potentials were measured using the conventional microelectrode technique from canine left ventricular papillary muscle and free-running Purkinje fibers, under normal and hypoxic conditions. Ionic currents were measured using the whole-cell configuration of the patch-clamp method. Acetylcholine at 5 μmol/L did not influence the action potential duration (APD) either in Purkinje fibers or in papillary muscle preparations. In contrast, it significantly lengthened the APD and suppressed the Purkinje–ventricle APD dispersion when it was administered after 5 μmol/L pinacidil application. Carbachol at 3 μmol/L reduced the pinacidil-activated I K-ATP under voltage-clamp conditions. Acetylcholine lengthened the ventricular action potential under simulated ischemia condition. In this study, we found that acetylcholine inhibits the I K-ATP and thus suppresses the ventricle–Purkinje APD dispersion. We conclude that parasympathetic tone may reduce the arrhythmogenic substrate exerting a complex antiarrhythmic mechanism during hypoxic conditions.

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug, which has recently been associated with increased cardiovascular risk, but its electrophysiological effects have not yet been properly studied in isolated cardiac preparations. We studied the effects of ibuprofen on action potential characteristics and several transmembrane ionic currents using the conventional microelectrode technique and the whole-cell configuration of the patch-clamp technique on cardiac preparations and enzymatically isolated ventricular myocytes. In dog (200 µM; n = 6) and rabbit (100 µM; n = 7) papillary muscles, ibuprofen moderately but significantly prolonged repolarization at 1 Hz stimulation frequency. In dog Purkinje fibers, repolarization was abbreviated and maximal rate of depolarization was depressed in a frequency-dependent manner. Levofloxacin (40 µM) alone did not alter repolarization, but augmented the ibuprofen-evoked repolarization lengthening in rabbit preparations (n = 7). In dog myocytes, ibuprofen (250 µM) did not significantly influence IK1, but decreased the amplitude of Ito and IKr potassium currents by 28.2% (60 mV) and 15.2% (20 mV), respectively. Ibuprofen also depressed INaL and ICa currents by 19.9% and 16.4%, respectively. We conclude that ibuprofen seems to be free from effects on action potential parameters at lower concentrations. However, at higher concentrations it may alter repolarization reserve, contributing to the observed proarrhythmic risk in patients.

The occurrence of members of the Pasteurellaceae and Neisseriaceae families was studied in dogs and cats. A total of 110 nasal and pharyngeal swab samples from 47 dogs and 8 cats were collected. Most of the strains were identified by 16S rDNA sequencing, except Frederiksenia canicola and Pasteurella multocida where species-specific polymerase chain reactions were applied. The most frequently isolated species was F. canicola, which occurred only in dogs, mainly in the pharyngeal cavity. The second commonest bacterium, P. multocida was found in both types of samples and in both hosts. Other species from the family Pasteurellaceae, such as Haemophilus haemoglobinophilus, Pasteurella canis and P. dagmatis, were detected only in dogs. All isolated species belonging to the family Neisseriaceae, mainly representing Neisseria weaveri, were found only in the pharyngeal cavity. Neisseria weaveri and N. zoodegmatis could be detected in both hosts. Neisseria dumasiana and N. canis were isolated from dogs, while N. shayeganii only from a cat. For phylogenetic analysis, rpoB gene sequencing was performed, where the strains were on monophyletic branches and clearly separated from each other. In this study, recently described species such as F. canicola, N. shayeganii and N. dumasiana were detected that had never been isolated in Hungary before.

Vagal nerve stimulation (VNS) has a meaningful basis as a potentially effective treatment for heart failure with reduced ejection fraction. There is an ongoing VNS randomized study, and four studies are completed. However, relatively little is known about the effect of acetylcholine (ACh) on repolarization in human ventricular cardiomyocytes, as well as the effect of ACh on the rapid component of the delayed rectifier K

Since 2010, outbreaks of haemorrhagic septicaemia (HS) caused by Pasteurella (P.) multocida capsular type B (PmB) emerged in Germany. In 2017, we noticed a close spatiotemporal relationship between HS outbreak sites and wolf (Canis lupus) territories. Thus, the main objectives of our study were to investigate the molecular epidemiology of German PmB-HS-isolates and to assess the role of wolves as putative vectors of this pathogen. We collected 83 PmB isolates from HS outbreaks that occurred between 2010 and 2019 and sampled 150 wolves, which were found dead in the years 2017 to 2019, revealing another three PmB isolates. A maximum-likelihood-based phylogeny of the core genomes of 65 PmB-HS-isolates and the three PmB-wolf-isolates showed high relatedness. Furthermore, all belonged to capsular:LPS:MLST genotype B:L2:ST122RIRDC and showed highly similar virulence gene profiles, but clustered separately from 35 global ST122RIRDC strains. Our data revealed that German HS outbreaks were caused by a distinct genomic lineage of PmB-ST122 strains, hinting towards an independent, ongoing epidemiologic event. We demonstrated for the first time, that carnivores, i.e., wolves, might harbour PmB as a part of their oropharyngeal microbiota. Furthermore, the results of our study imply that wolves can carry the pathogen over long distances, indicating a major role of that animal species in the ongoing epidemiological event of HS in Germany.

This study describes the prevalence of capsule biosynthesis genes, LPS genotypes, virulence associated genes and the analysis of the outer membrane protein (ompA) sequence of Pasteurella multocida isolates (n = 180) from different locations in Hungary, from various host species, including humans. When combining capsular types with LPS genotypes, eight capsule - LPS genotype combinations were detected. A: L3 was the most dominant in bovine and porcine isolates, A: L1 in feline and human isolates, while D: L3 was the most common among strains from small ruminants. The P. multocida toxin encoding gene toxA was highly prevalent among small ruminant and porcine strains, while in human, feline and bovine isolates it could not be detected. Combination of the tested virulence associated genes (hgbA, nanH, hgbB, tbpA, pfhA, hsf1, hsf2, tadD, ptfA) classified our P. multocida isolates into 13 different virulence gene profiles (VGPs). These VGPs showed an association with host species. Analysis of the ompA sequence data confirmed this distribution by host species, which may indicate that host adaptation is taking place. The typing scheme used in this study may be useful in epidemiological investigations.

African swine fever virus (ASFV) is an important animal pathogen causing substantial economic losses in the swine industry globally. At present, little is known about the molecular biology of ASFV, including its transcriptome organization. In this study, we applied cutting-edge sequencing approaches, namely the Illumina short-read sequencing (SRS) and the Oxford Nanopore Technologies long-read sequencing (LRS) techniques, together with several library preparation chemistries to analyze the ASFV dynamic transcriptome. SRS can generate a large amount of high-precision sequencing reads, but it is inefficient for identifying long RNA molecules, transcript isoforms and overlapping transcripts. LRS can overcome these limitations, but this approach also has shortcomings, such as its high error rate and the low coverage. Amplification-based LRS techniques produce relatively high read counts but also high levels of spurious transcripts, whereas the non-amplified cDNA and direct RNA sequencing techniques are more precise but achieve lower throughput. The drawbacks of the various technologies can be circumvented by the combined use of these approaches.

In this paper we report the phenotypic and partial genetic characterisation of a novel bacterium strain isolated from a cat with severe nephritis. Multilocus sequence analysis was performed on the 16S rRNA and three housekeeping (recN, rpoB, infB) gene sequences obtained by PCR. In accordance with the results of phenotypic tests, the phylogenetic analyses confirmed the relatedness of the new strain (6036) to the family Pasteurellaceae. On the phylogenetic trees, strain 6036 appeared in a separate branch, closest to that of the type species (Frederiksenia canicola) of the genus Frederiksenia. These two bacteria shared 95.14 and 76.88% identity in their partial 16S rRNA and recN gene sequences, respectively. The rpoB- and infB-based phylogenetic analyses indicated that strain 6036 is most closely related to Bibersteinia trehalosi (with 90.58% identity) and [Haemophilus] felis ATCC 49733 (89.50% identity), respectively. The predicted genome identity values, based on the recN gene sequences, suggested that strain 6036 can be classified into the genus Frederiksenia as a novel species. A PCR method, specific to strain 6036, was developed to allow its rapid and accurate identification and differentiation from F. canicola and other species of Pasteurellaceae. The minimal inhibitory concentrations of 18 antimicrobial agents for strain 6036 were also determined.

Riemerella anatipestifer causes anatipestifer disease in many avian species. A total of 185 R. anatipestifer strains isolated in Hungary between 2000 and 2014 from geese and ducks were tested against 13 antibiotics (ampicillin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, penicillin, spectinomycin, streptomycin, sulphamethoxazole—trimethoprim, sulphonamide compounds, and tetracycline) by the Kirby-Bauer disk diffusion method. The majority of the strains were susceptible to florfenicol (97.9%), ampicillin (95.1%), penicillin (93%), sulphamethoxazole—trimethoprim (92.4%), and spectinomycin (86.5%). The highest resistance rates were observed for flumequine, tetracycline, erythromycin and streptomycin (94%, 91.4%, 75.1% and 71.4% resistance, respectively). The resistance patterns showed some variation depending on the geographical origin of the strains. The average rate of extensive drug resistance was 30.3%, and its proportion tended to increase in the period examined.

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.

The possible interaction between Pasteurella multocida and the mycotoxin fumonisin B1 (FB1), recognised as one of the most often food/feed contaminant, was studied with the aim to evaluate whether and how FB1 can influence and/or complicate the development and severity of various pathological damages provoked by Pasteurella multocida in some internal organs of pigs. Heavier lung pathology was seen in pigs experimentally infected with Pasteurella multocida, when the same were exposed to 20ppm dietary levels of fumonisin B1 (FB1) as was assessed by gross pathology, pathomorphological examinations, clinical biochemistry and some immunological investigations. The most typical damages in FB1 treated pigs were the strong oedema in the lung and the slight oedema in the other internal organs and mild degenerative changes in the kidneys, whereas the typical pathomorphological findings in pigs infected with Pasteurella multocida was broncho-interstitial pneumonia. FB1 was found to aggravate pneumonic changes provoked by P. multocida in the cranial lobes of the lung and to complicate pneumonic damages with interstitial oedema in the lung. No macroscopic damages were observed in the pigs infected only with Pasteurella multocida. It can be concluded that the feed intake of FB1 in pigs may complicate or exacerbate the course of P. multocida serotype A infection.

Anatipestifer disease is a contagious disease caused by Riemerella anatipestifer, affecting primarily ducks, geese and turkeys, and characterised by listlessness, diarrhoea, sneezing, nasal discharge, and nervous signs. Sporadically, it occurs in a wide range of other domesticated and wild birds as well. The incidence and characteristics of the disease seen in the three main host species are summarised based on birds submitted for routine laboratory investigation in Hungary over the period 2010–2014. The infection was diagnosed in a higher percentage in geese (9.9%) and ducks (7.5%). It occurred in 5-day-old to 17-week-old geese and 3- to 6.5-week-old ducks, respectively. The pathological lesions were comparable in these two species: enlarged spleen, serofibrinous pericarditis, perihepatitis, airsacculitis, catarrhal enteritis, subcutaneous oedema and hyperaemia over the cranium, mucopurulent exudate in the nasal cavity and occasionally pneumonia, conjunctivitis, purulent arthritis and caseous salpingitis. In some cases, R. anatipestifer produced only secondary lesions, which complicated other diseases such as circovirus infection, mycotoxicosis, mycoplasmosis, or Derzsy’s disease. In turkeys, the disease occurred rarely (0.5%) and at an older age (12 to 19 weeks). The lesions most frequently seen were purulent osteomyelitis of the cranium and seropurulent meningitis. Purulent osteomyelitis in the cranium caused by R. anatipestifer infection had not been reported in turkeys previously. To various extents, other local lesions such as serofibrinous pericarditis, airsacculitis, arthritis, and in one case septicaemia were also observed. The high incidence of the disease in waterfowl underlines the importance of appropriate treatment and prevention that should be based on accurate diagnosis and antimicrobial susceptibility testing, proper biosecurity and vaccination with regard to the serotype(s) present on the farm.

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

The emergence of simultaneous resistance to multiple classes of antibiotics presents an increasing threat. Plasmid-borne multiresistance and integrative conjugative elements have been reported in Pasteurella multocida. We report an alternative strategy for the development of multiresistance observed in a P. multocida strain (Pm238) isolated from calf pneumonia. We identified genes integrated into the chromosomal DNA without known integrative and conjugative elements. These genes conferred resistance to streptomycin (strA), tetracycline (tetB), chloramphenicol (catAIII), and sulphonamides (sulII). We also detected mutation in the quinolone-resistance-determining regions of parC. No plasmids could be isolated from strain Pm238. These results suggest that P. multocida can accumulate multiple resistance determinants on the chromosome as single genes.

Isolates of Pasteurella multocida recovered from infected humans (n = 15) were characterized by traditional and molecular microbiological methods and were compared with cat-derived strains (n = 5). The most prevalent subspecies among strains from human infections was P. multocida subsp. septica (80%), and nearly all isolates showed a similar combination of virulence-associated genes. MLST analysis classified the 20 P. multocida strains into 16 different sequence types, and we assigned 11 new sequence types (ST), however, only one of those (ST 334) was shared by two human and one cat isolates. P. multocida subsp. septica strains formed a distinct phylogenetic group within the species. The strains showed resistance to erythromycin, clindamycin and sulfamethoxazole, and with two exceptions, resistance to tilmicosin was also detected. Each strain was susceptible to ampicillin, streptomycin, gentamycin, tetracycline, doxycycline, cefazolin, cefpodoxime, chloramphenicol, florfenicol and enrofloxacin. Common characteristics (virulence profile and antibiotic sensitivity pattern) shared by strains isolated from humans and cats support the view that domestic cats may serve as a potential reservoir for P. multocida.

Riemerella anatipestifer (RA) is a widely distributed bacterial pathogen of birds responsible for remarkable losses to poultry production, especially among waterfowl. We characterized the genomic diversity of 166 field isolates of RA, collected from geese and ducks, using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR). The field strains and five reference strains showed 17 distinct patterns consisting of five to 12 bands ranging from approximately 150–1800bp. The majority of the strains belonged to two closely related ERIC-PCR types (A and B), while the other types contained only a few isolates each. There was no association between ERIC-PCR type and host species, place, or year of isolation; however the ERIC-PCR pattern was correlated with serotype for most isolates. The majority of serotype 1 strains (101/107) belonged to ERIC-PCR type A while the remaining six strains represented five different ERIC-PCR types (D, G, L, M, and O). Serotypes 1,7 and 7 corresponded to ERIC-PCR types B and C, respectively. Serotypes 2, 4, and 10 could be subdivided by ERIC-PCR revealing two to four patterns within each serotype. These results indicate that ERIC-PCR may be a suitable technique for the molecular identification of RA serotypes, and the detection of subtypes within certain serotypes may aid further epidemiological investigations. RESEARCH HIGHLIGHTSERIC-PCR analysis of field R. anatipestifer strains revealed 17 distinct patternsMost strains belonged to two closely related ERIC-PCR typesSerotype 1 was the most prevalent serotype representing 64.5% of the strainsERIC-PCR may be suitable for molecular identification of R. anatipestifer serotypes ERIC-PCR analysis of field R. anatipestifer strains revealed 17 distinct patterns Most strains belonged to two closely related ERIC-PCR types Serotype 1 was the most prevalent serotype representing 64.5% of the strains ERIC-PCR may be suitable for molecular identification of R. anatipestifer serotypes

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A-E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.

This paper reports an outbreak of haemorrhagic septicaemia caused by Pasteurella multocida B:2 in beef calves, a disease that has not been described in the Hungarian literature since 1943, and has not been reported to the World Organisation For Animal Health (OIE) since 1970. Acute haemorrhagic septicaemia was confirmed in beef calves on one small farm, and was suspected on two further nearby holdings with concomitant unexplained losses. The source of the infection could not be determined. Apart from a short duration of depression and loss of appetite, the affected calves developed characteristic distal limb oedema. Gross findings in two calves submitted for laboratory examinations included subcutaneous oedema and haemorrhages on serous membranes, and in one case severe pharyngeal lymph node enlargement was observed. Histological examinations revealed lesions characteristic of septicaemia. Moderate to large amounts of Pasteurella antigens were detected in all organs tested by immunohistochemistry. Two isolates of P. multocida (Pm240, Pm241) were cultured from these cases and examined in detail. These were identified as P. multocida ssp. multocida biovar 3. Both were toxA negative and belonged to serotype B:2. Multilocus sequence typing was used to assign these to a new sequence type (ST64) that is closely related to other haemorrhagic septicaemia causing strains of P. multocida regardless of the host.

Background Two main genetic groups (B.12 and B.FTNF002-00) of Francisella tularensis ssp. holarctica are endemic in Europe. The B.FTNF002-00 group proved to be dominant in Western European countries, while strains of the B.12 group were isolated mainly in Northern, Central and Eastern Europe. The clinical course of tularemia in the European brown hare (Lepus europaeus) also shows distinct patterns according to the geographical area. Acute course of the disease is observed in hares in Western European countries, while signs of sub-acute or chronic infection are more frequently detected in the eastern part of the continent. The aim of the present study was to examine whether there is any difference in the virulence of the strains belonging to the B.FTNF002-00 and B.12 genetic clades. Results Experimental infection of Fischer 344 rats was performed by intra-peritoneal injection of three dilutions of a Hungarian (B.12 genotype) and an Italian (B.FTNF002-00 genotype) F. tularensis ssp. holarctica strain. Moderate difference was observed in the virulence of the two genotypes. Significant differences were observed in total weight loss values and scores of clinical signs between the two genotypes with more rats succumbing to tularemia in groups infected with the B.FTNF002-00 genotype. Conclusions Results of the experimental infection are consistent with previous clinical observations and pathological studies suggesting that F. tularensis ssp. holarctica genotype B.FTNF002-00 has higher pathogenic potential than the B.12 genotype. Electronic supplementary material The online version of this article (doi:10.1186/s12917-017-0968-9) contains supplementary material, which is available to authorized users.

Bordetella bronchiseptica is a widespread Gram-negative pathogen occurring in different mammal species. It is known to play a role in the aetiology of infectious atrophic rhinitis of swine, canine kennel cough, respiratory syndromes of cats, rabbits and guinea pigs, and sporadic human cases have also been reported. In this study, 93 B. bronchiseptica strains were examined from a broad range of host species and different geographical regions using restriction fragment length polymorphism analysis of polymerase chain reaction products of flaA to reveal the possible host-specificity of the flagellin. Eight types (A-H) of flaA were identified, including five newly described ones (D-H). All but one of the 22 B. bronchiseptica strains from swine showed type B fragment pattern. The eighteen Hungarian isolates of canine origin were uniform (type A) while in other countries type B and D were also present in dogs. The sequence and phylogenetic analysis of 36 representative strains of flaA types revealed four clusters. These clusters correlated with flaA PCR-RFLP types and host species, especially in pigs and dogs. The revealed diversity of the strains isolated from human cases indicated possible zoonotic transmissions from various animal sources.

A more complicated pathology was observed in female pigs infected with Mycoplasma hyopneumoniae, when the same were exposed to 20 ppm dietary levels of fumonisin B1 (FB1) starting 14 days before infection for a period of 42 days as was assessed by gross pathology and pathomorphological examinations or computed tomography, and also manifested by the strong deterioration of the pneumonic process in two pigs and the subsequent euthanizing of one pig. Typical damages in FB1-fed pigs were a strong oedema in the lung and slight oedema in the other internal organs and mild degenerative changes in the kidneys, whereas the typical pathomorphological changes in M. hyopneumoniae-infected pigs corresponded to the morphologic pattern of a catarrhal bronchointerstitial pneumonia more pronounced in the cranial and middle lobes or in the cranial third of the caudal lobe of the lung. The pigs treated by both pathogens (toxic and infectious) revealed strong oedematous changes in the interstitium of lung in addition to deteriorated and extended bronchointerstitial pneumonic process.

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

Mycoplasma hyopneumoniae has a primary role in the porcine respiratory disease complex (PRDC). The objective of this study was to determine whether fumonisin mycotoxins influence the character and/or the severity of pathological processes induced in the lungs of pigs by Mycoplasma hyopneumoniae. Four groups of pigs (n = 7/group) were used, one fed 20 ppm fumonisin B1 (FB1) from 16 days of age (group F), one only infected with M. hyopneumoniae on study day 30 (group M), and a group fed FB1 and infected with M. hyopneumoniae (group MF), along with an untreated control group (group C). Computed tomography (CT) scans of infected pigs (M and MF) on study day 44 demonstrated lesions extending to the cranial and middle or in the cranial third of the caudal lobe of the lungs. The CT images obtained on study day 58 showed similar but milder lesions in 5 animals from group M, whereas lungs from 2 pigs in group MF appeared progressively worse. The evolution of average pulmonary density calculated from combined pixel frequency values, as measured by quantitative CT, was significantly influenced by the treatment and the age of the animals. The most characteristic histopathologic lesion in FB1-treated pigs was pulmonary edema, whereas the pathomorphological changes in Mycoplasma-infected pigs were consistent with catarrhal bronchointerstitial pneumonia. FB1 aggravated the progression of infection, as demonstrated by severe illness requiring euthanasia observed in 1 pig and evidence of progressive pathology in 2 pigs (group MF) between study days 44 and 58.

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.

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.

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.

A rapid, easy method involving a polymerase chain reaction (PCR) assay followed by restriction fragment length polymorphism (RFLP) analysis was developed to differentiate the 2 phylogenetic lineages of Pasteurella multocida. The PCR targeted the 16S ribosomal RNA gene, and the RFLP involved separate digestions with HindIII, EarI, and MlsI. The method was applied to 16 isolates of P. multocida from different hosts and the isolates were clearly assigned to 1 of the 2 lineages. The phylogenetic lineages did not match with the phenotypic-based identification at the subspecies level.

The zoonotic impact of Pasteurella species in human wounds caused by cats and dogs has increased recently. In this study, the effectiveness of the Biolog Microstation ID System (Biolog, Hayward, CA) for the identification of certain species of Pasteurella sensu stricto was analysed. Thirty-eight isolates originating from dogs and cats were studied by pheno- and genotypic methods. The classical biochemical tests identified these isolates as P. multocida, P. dagmatis, and P. canis, while the Biolog system distinguished only 2 categories: P. multocida and P. dagmatis. The sodA gene sequence-based phylogenetic analysis revealed that the isolates identified as P. dagmatis by the Biolog system were either P. dagmatis, P. canis, or P. dagmatis-like genomospecies. The low discrimination power of the Biolog system in the case of these closely related Pasteurella species draws attention to the need of continuously improving the database of automated microbial identification systems.

Four urease-negativeBordetella bronchisepticaisolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs™ assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific andureC PCR, and all strains showed uniform bands in PCR-RFLP studies offlaA genes. The reason for the lack of urease activity, a characteristic considered speciesspecific forB. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification ofB. bronchisepticaseems questionable.

Three Pasteurella dagmatis-like strains recovered from the feline oral cavity were analysed by traditional biochemical tests, the Biolog Microstation™ ID System, and 16S rRNA and sodA gene sequence analysis. The molecular biological methods revealed that these strains differ from P. dagmatis, forming a new genomospecies in the genus Pasteurella sensu stricto. Furthermore, sequence analysis and multiple alignments of 16S rRNA and the sodA gene established that the P. pneumotropica NCTC10827 and the P. dagmatis-like strains described here possess high genetic similarity.

The antimicrobial susceptibility of 19 Bordetella avium and 36 Ornithobacterium rhinotracheale strains was tested by the Kirby-Bauer disk diffusion method, and the minimal inhibitory concentrations (MIC) of amoxicillin, doxycycline and erythromycin were also determined. Most O. rhinotracheale strains were resistant to nalidixic acid, sulphamethoxazole–trimethoprim and gentamicin, and were susceptible to ampicillin, chloramphenicol, spectinomycin and tilmicosin. All B. avium strains were resistant to ceftiofur and lincomycin and susceptible to doxycycline, gentamicin, polymyxin B, spectinomycin and sulphonamides. The MICs ranged widely for all three antibiotics tested against O. rhinotracheale strains, from 0.12 μg/ml to 32 μg/ml for amoxicillin and erythromycin, and from 0.6 μg/ml to 32 μg/ml for doxycycline. For B. avium isolates, the MIC values ranged from ≤ 0.03 μg/ml to 1 μg/ml for amoxicillin, from ≤ 0.03 μg/ml to 0.12 μg/ml for doxycycline and from 8 μg/ml to 16 μg/ml for erythromycin. These findings support the idea that the use of antibiotics in a region or a farm may affect antimicrobial resistance and underline the need for prudent application of antibiotic therapy based on proper antimicrobial susceptibility testing.

To the Editor: Francisella tularensis, the etiologic agent of tularemia, is a highly infectious zoonotic agent. F. tularensis subsp. holarctica (type B) is found throughout the Northern Hemisphere and is the only subspecies found in Europe (1). Lagomorphs and rodents probably serve as the primary mammalian reservoir hosts, and hematophagous arthropods, such as ticks, play a role as vectors and hosts (2,3). Although F. tularensis is a potential agent of biological warfare and several emergences and reemergences of tularemia have been reported around the world (1,4), the epizootiology of the disease is only partially understood. The aim of our study was to analyze factors that influence the emergence of tularemia in Hungary. The study area (15,475 km2) included 3 counties in eastern Hungary. The analyzed data represented a period of 25 years, March 1984–February 2010. Annual F. tularensis–specific seroprevalence data for the European brown hare (Lepus europaeus) population were obtained by slide agglutination testing during the winter (December and January) screening of 2,500–25,000 animals (Technical Appendix). Population density data (animals/km2) for hares were based on February line transect counts and were obtained from the Hungarian Game Management database (www.vvt.gau.hu/vadgazdalkodasi_statisztikak.htm). Common vole (Microtus arvalis) densities (calculated from the number of active burrows/hectare during November) for 1996–2010 were obtained from the Central Agriculture Office, Budapest, Hungary. Vole density was scaled from 0 (absent) to 10 (peak population). The annual number of tularemia cases in humans (based on clinical history and tube agglutination test results) was obtained from the National Center for Epidemiology, Budapest. The data were regrouped according to the yearly biologic cycle (March–February) for hares and voles (Figure), and relationships between these yearly data were quantified by using the Spearman rank correlation coefficient (5) at county and regional levels. A 2–3 year cycle was characteristic for the analyzed data. A significant positive correlation was found among the number of tularemia cases in humans and the seroprevalence of F. tularensis among European brown hares (Spearman ρ = 0.73; p

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.

Background Mycoplasma bovis is a worldwide pathogen, causative agent of pneumonia, mastitis, arthritis, and a variety of other symptoms in cattle. The economic losses due to mycoplasma pneumonia could be reduced by antibiotic treatment. The aim of the present study was to determine the in vitro susceptibility of M. bovis strains isolated from cattle in Hungary to eleven antibiotics. Results Minimal inhibitory concentration (MIC) values of 35 M. bovis strains collected from different parts of Hungary between 2010 and 2013 were determined by the microbroth dilution method. Strains with high MIC values were found in the case of all applied antibiotics. The most effective antibiotics tested in vitro were fluoroquinolones (MIC90 danofloxacin 0.312 μg/ml, enrofloxacin 0.312 μg/ml, marbofloxacin 0.625 μg/ml). Our results confirm the observations of increasing MIC values to antibiotics commonly used in the therapy of mycoplasma infections, primarily to tetracyclines; tetracycline (MIC90 16 μg/ml) and oxytetracycline (MIC90 ≥ 64 μg/ml) and macrolides; tylosin (MIC90 ≥ 128 μg/ml) and tilmicosin (MIC90 ≥ 128 μg/ml). The growth of many M. bovis strains was not inhibited by gentamicin (MIC90 8 μg/ml), spectinomycin (MIC90 ≥ 256 μg/ml), florfenicol (MIC90 8 μg/ml) or lincomycin (MIC90 ≥ 64 μg/ml). Conclusions Our results emphasize the necessity of periodic testing for antibiotic susceptibility in this geographic region. Based on our in vitro examinations, fluoroquinolones could be the most effective drugs for the therapy of M. bovis infections in Hungary. However, current antimicrobial use policies have to be taken into account to avoid further antibiotic resistance development and to reserve fluoroquinolones for the treatment of severe infections which have responded poorly to other classes of antimicrobials.

Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A–D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR–RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.