Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs.

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.

Rabbit haemorrhagic disease: an investigation of some properties of the virus and evaluation of an inactivated vaccine.

An inactivated vaccine against rabbit haemorrhagic disease (RHD), developed and tested in our laboratory, is produced commercially by Bioveta, Ivanovice, Czechoslovakia. Rabbits developed full protection against infection 3 weeks after the administration of a single dose. Antibodies were detectable from day 5 after vaccination. Naturally acquired antibodies were demonstrated in some rabbits kept on commercial farms. The virus survived at least 225 days in an organ suspension kept at 4 degrees C, at least 105 days in the dried state on cloth at room temperature (around 20 degrees C), and at least 2 days at 60 degrees C, both in organ suspension and in the dry state. Experimental infection of rabbits younger than 2 months was successful in some animals. Hares, guinea pigs, white mice, golden and Chinese hamsters, chinchillas and hysterectomy-derived, colostrum-deprived piglets were resistant to infection.

[Electron microscopy of Aujeszky's disease virus in explants of Gasser's ganglion from pigs with a latent infection]. / Elektronová mikroskopie viru Aujeszkyho choroby (ACH) v explantátech Gasserova ganglia z prasat s latentní infekcí.

Different developmental stages of the Aujeszky's disease virus were demonstrated by electron microscopy in the ultra-thin slices by the cultivated fragments of the Gasserian ganglion (G. g.) of two pigs latently infected with the Aujeszky's disease virus (ADV). In a pig vaccinated with the inactivated vaccine against the disease, the virus was detected in the G. g. cells 186 days after virus challenge, the reactivation of latency being obtained after immunosuppression with dexamethasone. In the non-vaccinated pig the virus was detected in G. g. cells after three months from experimental infection. In the ultra-thin slices the largest amount of virus was located in the nuclei and cytoplasm of satellite and Schwann's cells, in the connective-tissue cells and in the extracellular space. In the ganglion cells the virus was present in the cytoplasm and sporadically in the myelinized axons.

[The effect of short-term isolation of vaccinated chicks on the incidence of Marek's disease]. / Vliv krátkodobé izolace vakcinovaných kurat na výskyt Markovy choroby.

In chickens immunized by normal and tenfold doses of MARVAK vaccine (100 and 1,000 PFU) we investigated protective effects against the natural contact infection of chickens that were exposed to the infection immediately after vaccination, or at the intervals of 48 and 72 hours, and 7 and 14 days. Studying the elimination of Marek's disease virus by feather follicles, the health condition of chickens, tumor frequency and mortality rate we demonstrated that neither of these doses could protect the chickens from the disease if they were exposed to contact infection just after vaccination. An expressive protective effect was observed in chickens isolated for 7-14 days and there was not recorded any difference in the protection against the disease after the doses of 100 and 1,000 PFU. The main and feasible measure against MD is to prevent an early infection of newborn chickens by improving the zoohygienic conditions.

[Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period]. / Vyuzití enzymoimunologické (ELISA) diagnostiky Aujeszkyho choroby prasat pri ozdravování chovu.

Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation.

[The present epizootological situation in intensively bred cattle in relation to the morbidity and mortality of calves during their rearing]. / Soucasná epizootologická situace v intenzívních chovech skotu v souvislosti s nemocností a mortalitou telat v prubehu jejich odchovu.

The rise and mass incidence of intestinal and respiratory infections in calves kept in large herds depend on a joint influence of infective agents and numerous endogenous and exogenous factors, mostly with non-specific action. Therefore, purely medical approach fails to provide efficient prevention. Detailed epizootological analysis is needed for taking actual measures; the analysis should concern potential respiratory and intestinal pathogens and should also cover the persistent infections such as IBR, BVD-MD and others. The immunological profile of herd, very unfavorable with the high culling rate, will improve as a result of the introduction of a purpose-oriented vaccination programme, higher level of colostral nutrition, and separate rearing of calves, isolated from the dams, in calf houses managed by the all-in-all-out system.

[Survival of Aujeszky's disease virus in infected pig slurry]. / Prezívání viru Aujeszkyho choroby (ACH) v infikované prasecí kejde.

It has been demonstrated that after experimental infection of pig slurry from the space under the slatted floor (infection dose of 10(6)PFU per ml), the Aujeszky's disease virus (ADV) survived for 72 hours at the temperature of 15 degrees C and at pH 6.5, but was inactivated after 96 hours. When technologically treated pig slurry from the storage tanks was saturated with water and infected with ADV at the dose of 10(5)PFU per ml, the virus survived for 23 days when kept at 15 degrees C and 4 degrees C and at pH 6.8, but was inactivated under the same conditions after 30 days. When the infective ADV dose in the technologically treated pig slurry in the storage tanks was reduced to 10(4)PFU per ml, the virus survived 16 days at +4 degrees C and pH 7.0 and 8.0 but was inactivated within 23 days after infection.

[The effect of colostral immunity on the active formation of antibodies in pigs after the administration of inactivated vaccine against Aujeszky's disease]. / Vliv kolostrální imunity na aktivní tvorbu protilátek u prasat po aplikaci inaktivované vakcíny proti Aujeszkyho chorobe (ACH).

In a large herd of pigs where a trial was performed to cure the animals from Aujeszky's disease (AD) by applying to all animals an inactivated vaccine, a post-vaccination antibody response was studied in piglets coming from the sows that were vaccinated several times. When the piglets were vaccinated at the age of eight weeks (the average virus-neutralizing titer (VNT) of colostral antibodies was 1:11.4) and revaccinated at the age of 11 weeks, 73% of the forty-five animals (examined at the age of 17 weeks) did not have any virus-neutralizing (VN) antibodies in the blood serum. After the third vaccination dose (at the age of 17 weeks), 11% of piglets did not have any VN antibodies if they were examined at the age of 22 weeks (the average antibody VNT was 1:15.3). Applying the ELISA procedure, the antibodies were demonstrated in the sera of all piglets after three vaccination doses. Shifting the time intervals of vaccination (at the age of 8, 13 and 19 weeks), the VN antibodies were found out after three vaccination doses in the sera of all piglets examined at the age of 23 weeks (the average VNT was 1:56.4). After three vaccination doses at the age of 12, 17 and 23 weeks, the VN antibodies were also demonstrated in all piglets at the age of 27 weeks (the average VNT was 1:208).

Experimental transmission and electron microscopic demonstration of the virus of haemorrhagic disease of rabbits in Czechoslovakia.

Intramuscular administration of the filtrate of organ suspensions, prepared from a dead rabbit, killed 62.9% of inoculated rabbits within 1 to 5 days, while 93.3% died after intranasal administration of the same inoculum. The virus survived freeze-drying and was resistant to treatment with 0.4% formaldehyde when incubated at 37 degrees C for 1 hour and 4 degrees C for the subsequent 12 hours, but lost its infectivity when the treatment was prolonged to 3 hours at 37 degrees C and 3 days at room temperature. Its infectivity was also inhibited by reconvalescent serum. The virus could not be detected after 3 passages in primary rabbit kidney cell cultures. Electron microscopy of negatively stained preparations demonstrated icosahedral virus particles with a diameter of 29 to 33 nm without an envelope. Accurate morphological classification has not yet been completed. Incubation with a reconvalescent serum, diluted 1:20 or 1:40, resulted in the formation of immune complexes, detectable by electron microscopy.

Enzyme-linked immunosorbent assay of antibodies to rabbit haemorrhagic disease virus and determination of its major structural proteins.

An ELISA was developed for the determination of antibodies to rabbit haemorrhagic disease virus (RHDV) in whole blood and blood serum of rabbits. Naturally acquired antibodies were detected in 19.4% of blood samples collected from 1461 rabbits in 43 farms apparently free of the disease, 19.7% samples were doubtful and 60.9% of the rabbits were free of antibodies to RHDV. Their presence has a considerable effect on the resistance of rabbits to infection with RHDV. Antibodies were also found in rabbit blood serum samples collected up to 12 years before the first outbreaks of RHD were reported. Up to 14 viral protein antigens were determined by PAGE and Western blot analysis, of which three with Mr values of 61K, 38K and 52K were major proteins, the 61K being dominant. Our hyperimmune sera, a Chinese reference serum and sera with positive antibody titres, including those collected several years before the first outbreaks of RHD, reacted identically with these antigens in the Western blot analysis. The data obtained suggest that naturally acquired antibodies are a product of a specific response to prior infection with an avirulent strain of the virus.