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Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

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AIMS: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease. Genotypic discrimination of MAP isolates is pivotal to epidemiological studies requisite for revealing infection sources and disease transmission. This study was undertaken to determine the genetic diversity of MAP strains from diverse sources. METHODS AND RESULTS: Five hundred and sixty-nine MAP isolates were collected during an 8-year period from nine animal species, originating from seven European countries, including the whole geographic region of Hungary. Isolates were classified into cattle type and sheep type, and 515 strains were included in mycobacterial interspersed repetitive units-variable-number tandem repeat analysis. CONCLUSIONS: The same genotype was found in different host species cohabiting on the same property, demonstrating interspecies transmission. Detecting identical patterns in numerous related animals underlines the importance of vertical transmission. The revealed 15 genotypes expose relatively low strain diversity and indicate the need of an improved typing system that provides higher resolution in the case of this subspecies. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results demonstrate the circulation and transmission of different MAP strain types among individuals, herds and even wildlife reservoirs in Hungary and other European countries; correlation between production type or breed and MAP genotype is hypothesized.

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We investigated a Q fever outbreak with human patients showing high fever, respiratory tract symptoms, headache and retrosternal pain in southern Hungary in the spring and summer of 2013. Seventy human cases were confirmed by analysing their serum and blood samples with micro-immunofluorescence test and real-time PCR. The source of infection was a merino sheep flock of 450 ewes, in which 44.6% (25/56) seropositivity was detected by enzyme-linked immunosorbent assay. Coxiella burnetii DNA was detected by real-time PCR in the milk of four of 20 individuals and in two thirds (41/65) of the manure samples. The multispacer sequence typing examination of C. burnetii DNA revealed sequence type 18 in one human sample and two manure samples from the sheep flock. The multilocus variable-number tandem repeat analysis pattern of the sheep and human strains were also almost identical, 4/5-9-3-3-0-5 (Ms23-Ms24-Ms27-Ms28-Ms33-Ms34). It is hypothesised that dried manure and maternal fluid contaminated with C. burnetii was dispersed by the wind from the sheep farm towards the local inhabitants. The manure was eliminated in June and the farm was disinfected in July. The outbreak ended at the end of July 2013.

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The European brown hare (Lepus europaeus) is an important reservoir of Brucella suis biovar 2 and also of the life-threatening zoonotic agent Francisella tularensis. Since both bacteria can produce similar gross pathological lesions in this species, laboratory tests are necessary for the final diagnosis. The aim of the present study was to develop an immunohistochemical method for the detection of B. suis infection and to describe the pathological and histological lesions caused by B. suis in European brown hares. Hyperimmune serum for immunohistochemistry (IHC) was produced by subcutaneous infection of mice with 2 × 10(9) colony forming units of live B. suis biovar 2, injected four times at 1-week intervals. The antiserum did not react with F. tularensis or Yersinia pseudotuberculosis in IHC and displayed only weak cross-reaction with B. canis. Numerous, yellow-white necrotic foci (0.1-0.5 cm diameter) were found in the spleen of five B. suis-infected female European brown hares and also in the lung, uterus, kidney or liver of four of these cases. Microscopically, the foci comprised single or coalescing granulomas with a central necrotic area. Both bacterial isolation and IHC gave positive results for B. suis infection in these animals. B. suis antigens were found as granular or amorphous extracellular material in the necrotic centre of several granulomas. IHC appears to be a suitable complementary diagnostic method for the detection of B. suis infection in the European brown hare.

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The European brown hare (Lepus europaeus) plays an important role in the ecology of tularemia, and it may serve as a significant source of human infection. The aim of the present study was to examine the lesions induced by Francisella tularensis in 50 cases of naturally infected seropositive European brown hares. Gross pathological examination revealed scant to numerous grayish-white foci with diameters of 0.1 to 1.0 cm in single organs (24 cases) or multiple organs (20 cases) in 44 of 50 cases (88%). These lesions proved to be areas of granulomatous inflammation, frequently encompassing necrosis. F tularensis antigen was detected with immunohistochemistry in 46 of 50 cases (92%), whereas F tularensis ssp holarctica was isolated by culture and identified by polymerase chain reaction from 35 of 50 cases (70%). Infection by the respiratory route is suggested by the presence of the tissue lesions in thoracic organs of 44 of 50 cases (88%). These results emphasize the importance of the European brown hare as a reservoir of tularemia.

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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.

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Dicynone has been in use in all premature births prophylactically since 1987 by the authors. The administration of the drug begins before or during delivery. The diagnoses of cerebral haemorrhage was established on autopsy and the cases were compared with the previous years when Dicynone was not administered. During prophylactic use of Dicynone the cerebral haemorrhages significantly reduced among premature babies. It is well known, that the etiology of the cerebral haemorrhages are multifactorial. Their favourable experiences confirm the literary communications, whereas use of Dicynone can be one of the efficacious preventive drug against palsy of the premature babies.

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