Detection of rabbit haemorrhagic disease virus 2 (GI.2) in Poland.

In this paper we present the first cases of rabbit haemorrhagic disease virus 2 (RHDV2 - GI.2) in Poland. The virus was detected in liver samples of RHD-suspected rabbits from Lodzkie and west Pomeranian voivodeships. In both cases, the typical clinical symptoms of the disease were observed despite the fact that the rabbits were previously vaccinated against RHD. In order to extend the analysis of the RHDV2 strain infecting the rabbits, the entire VP60 and NSP genes were amplified and sequenced. The results of rRT-PCR assay have shown that tested RHDV samples were positive for the presence of RHDV2. In the phylogenetic analysis of vp60gene the first Polish RHDV isolates (RED 2016 and VMS 2017) clustered together with the reference RHDV2, meaning they represent new evolutionary RHDV linkeages. The first Polish RHDV2 isolates showed about 97% nucleotide sequence identity with the reference RHDV2 strains and approximately 18% difference from classic RHDV and RHDVa variants.

Serological Survey for RHD Antibodies in Rabbits from Two Types of Rabbit Breeding Farms.

Seroprevalence studies of RHDV antibodies in domestic rabbits were conducted between 2008-2014. A total of 12,169 sera from the provinces of central, southern and south-east Poland, including 7,570 samples collected from mixed-breed rabbits reared in smallholder farms and nearly 4,600 sera taken mainly from unvaccinated rabbits kept in industrial farms, were examined using ELISA tests. Additionally, cross-reactivity of selected tested and control archival sera using both classic RHDV and RHDVa antigens was determined by HI assay. The overall seroprevalence was 13.3%. In rabbits with unkown history of immunisation or RHD infection which came from small farms, RHDV antibodies were detected in 6.1% ranging between 1.0% to 17.2% of animals. In rabbits of the same group, but with a declared vaccination status, or confirmed exposure to an infectious virus, or coming from exposed females, the seroprevalence ranged from 83% to 100%. Among unvaccinated meat rabbits aged 71 to 90 days from industrial farms, low (1.85%, 4.17%, 11%), medium (34%, 54%) or high rates (98.7%) of seropositivity were detected. The seroconversion recorded in adult vaccinated females from industrial farms was 70% and 95%. Generally, the antibody levels examined by ELISAs and HI were comparable. However, a number of sera from the rabbits from small farms, as well as archival sera, showed clear differences. Several-fold differences in antibody titers, evidenced mainly in the postoutbreak sera, indictaed the contact of animals with RHDVa antigen. The overall results of the survey revealed a great proportion of seronegative rabbits potentially highly susceptible to RHD infection. In combination with the emergence of a novel pathogenic RHD virus type (RHDV2), it poses a severe risk of a next wave of fatal disease cases spreading in the native population of domestic rabbits, especially in farms with a traditional system of husbandry.

Identification of Polish RHDVa subtype strains based on the analysis of a highly variable part of VP60 gene.

In order to determine the genetic variability of Polish RHD virus strains and to confirm the presence of genetic variant (RHDVa) subtype the partial nucleotide sequences of capsid protein gene, including two highly variable regions C and E, were examined. Phylogenetic analyses of 15 viral strains obtained over 18 years revealed the presence of three genetic groups. The oldest RHDV strains exhibit very close amino acid sequence similarity (98-99%) to the German FRG89 reference strain and most of European strains of the same period, as well as Chinese isolate from 1984. The HA-negative strains and isolates with variable reactivity in the HA test belong to the second subgroup and exhibit an intermediate level of variability (about 3%) in the analysed VP60 gene fragment. The most genetically variable strains (6-7%) clustered to RHDVa subtype. The analysis of nucleotides and amino acid sequences demonstrated three pairs of well conserved RHDV strains, isolated over 3, 6 and 10-year period.

beta-agonistic bronchodilators: comparison of dose/response in working rat hearts.

STUDY OBJECTIVES: Different beta-agonists are compared with regard to their cardiodepressive side effects. DESIGN: The metaphenolic bronchodilators reproterol, salbutamol, fenoterol, and terbutaline were introduced at a dosage of 0.0005 micromol to a maximum of 10 micromol per gram of heart tissue into the isolated working rat heart under hypoxic conditions, and the response was observed during subsequent reoxygenation. As an index of external heart work, aortic flow was measured. Heart rate, coronary flow, and developed pressure were recorded. At the end of heart perfusion, mitochondria were isolated and analyzed for adenosine triphosphatase activity, adenosine triphosphate (ATP) synthesis, and membrane fluidity. Moreover, intact mitochondria and lipid peroxidation were investigated using a model system. MEASUREMENTS AND RESULTS: Compared to controls, reproterol gave the most favorable results, with an increase of 25 to 30% of aortic flow during reoxygenation at a concentration of 10 micromol/g heart tissue. In contrast, both fenoterol and salbutamol at a concentration of 1 micromol/g heart tissue decreased aortic flow during reoxygenation, whereas terbutaline had a negative influence on aortic flow at 0.01 to 0.1 micromol/g heart tissue. Mitochondria of these hearts were isolated at the end of the experiment. Mitochondrial ATP synthesis was increased above controls at nearly all concentrations of reproterol. ATP synthesis was decreased at 1 micromol and 10 micromol fenoterol. As little as 0.0005 micromol terbutaline decreased ATP synthesis by 50%. In intact mitochondria, adenosine diphosphate (ADP) to oxygen ratios were found to be increased with terbutaline and fenoterol, indicating ADP consumption by myokinase activation. Lipid peroxidation was increased in a model system between concentrations of 0.002 micromol/mg and 0.04 micromol/mg phosphatidylcholine by fenoterol and terbutaline, whereas a decrease was noted with reproterol. Membrane fluidity was found increased after addition of reproterol, which supports the evidence of efficient ATP synthesis by this compound. CONCLUSIONS: Cardiodepressive side effects and greater toxicity of fenoterol and terbutaline were found under the conditions of our experiment. Salbutamol and, in particular, reproterol appear much better tolerated. In addition to partial beta-adrenergic agonism, reproterol may exert an inhibitory influence on adenosine receptor sites and phosphodiesterase, which could result in membrane stabilization by saving cyclic adenosine monophosphate or ATP.

A new variant of the viral haemorrhagic disease of rabbits virus.

A new variant of viral haemorrhagic disease of rabbits (VHD) virus, recently detected in Poland and called Blaszki (BLA), gives positive results in enzyme-linked immunosorbent assay (ELISA) and exhibits viral protein of 60 kilodaltons (VP 60), as detected by Western blot analysis. This BLA variant of VHD virus has caused high morbidity and mortality in rabbits, as have other reported variants with similar clinical signs and pathological lesions, but-in contrast to other variants-the BLA variant gave negative results in the haemagglutination test. This development indicates the limitations of haemagglutination testing in the diagnosis of VHD.

[Isolation and some properties of restriction endonuclease from Bacillus amyloliquefaciens]. / Vydelenie i nekotorye svoistva restriktsionnoi endonukleazy ia Bacillus amyloliquefaciens.

A partially purified preparation of restriction endonuclease Bam I was isolated from the cells of Bacillus amyloliquefaciens. The purification procedure was a modification of a method described by Wilson and Young. Isolation and purification of the enzyme involved disruption of the cells by ultrasonication, treatment with streptomycin sulfate, fractionation by ammonium sulfate, chromatography on DEAE-cellulose and hydroxylapatite and rechromatography on DEAE-cellulose. Restrictase Bam I splits the linear double-chain DNA molecule of phage gamma into six fragments. The enzyme retained its stability under storage on the ice for 1,5--2 months in a Na- or K-phosphate buffer with beta-mercaptoethanol (10 mM).