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.

Bluetongue in Europe and the role of wildlife in the epidemiology of disease.

The article reviews a current bluetongue (BT) epidemiological situation in Europe, BT restricted zones and the role of wild ungulates as a reservoir for bluetongue virus (BTV) and its transmission. BT has been eradicated from central and northern Europe, however it is still circulating in some regions of southern and south-eastern Europe. According to the recent information of the Directoriate General for Health and Consumer Affairs (DG SANCO) disease caused by BTV1 was spreading at the beginning of 2014 in Corsica (France). Moreover, four BTV1 cases were noticed in the west Spain (Cáceres province), 59 BTV4 outbreaks in south Spain (Andalusia), 10 in the region of Algarve in Portugal and about 200 outbreaks of BTV4 in Greece (Peloponesse and Evros regions). On 4th July the first outbreak of BTV4 was also confirmed at the south Bulgarian border and by 5th September 2014 disease was noticed in 21 of 28 administrative districts of Bulgaria. In August 2014 the BTV4 disease was reported in south-east of Romania and as for 8th September 184 outbreaks of BT were confirmed in 17 counties of this country. As of 3 September 2014 in Europe there has been fourteen BT-affected zones, in different regions of Italy, Spain, Portugal, Cyprus, Malta, France (Corsica), Greece, Bulgaria and Romania. Most species of wild ruminants and camelids are susceptible to BTV infection, although frequently asymptomatically. Wild sheep, bighorn and mouflon, are susceptible to BTV infection and can develop fatal clinical disease, as do domestic sheep. Experimental or natural infection of antelope, wapiti, musk, ox, bison, yak, white-tailed deer and African buffalo also produced clinical disease, whereas blesbock, mountain gazelle, roe deer, red deer and Eurasian elk did not show clinical sign after natural or experimental infection and infection was recognized by the presence of BTV viral RNA or specific antibodies. The wildlife due to the long-term carrier state may act as a reservoir for BTV and play an important role in its transmission.

The evolution of bluetongue virus: genetic and phenotypic diversity of field strains.

Bluetongue virus (BTV), the aetiological agent of bluetongue (BT), is a small (about 70 nm in diameter) icosahedral virus with a genome composed of ten linear segments of double-stranded RNA (dsRNA), which is packaged within an icosahedral nucleocapsid composed of seven structural proteins. The BTV genome evolves rapidly via genetic drift, reassortment of genome segments (genetic shift) and intragenic recombination. This evolution, and random fixation of quasispecies variants during transmission of BTV between susceptible animals and vectors appear to be the main mechanism leading to the observed genetic diversity amongst BTV field strains. The individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Reassortment of BTV genes is responsible for genetic shift among strains of BTV, and has been demonstrated after infection of either the ruminant host or insect vector with different strains or serotypes of BTV. Intragenetic recombination, whereby mosaic genes are generated from the "splicing" together of homologous genes from different ancestral viral strains, has been demonstrated for BTV. The genetic variation of BTV is likely responsible for differences in the virulence and other phenotypic properties of individual field strains of the virus.

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.

Bluetongue vaccines in Europe.

The article reviews the history, present status and the future of BT vaccines in Europe. So far, an attenuated (modified live viruses, MLV) and inactivated virus vaccines against BT were developed and used in the field. Moreover, the virus-like particles (VLPs) produced from recombinant baculovirus, and live recombinant vaccinia or canarypox virus-vectored vaccines were tested in the laboratory. The main aims of BT vaccination strategy are: to prevent clinical disease, to reduce the spread of the BTV in the environment and to protect movement of susceptible animals between affected and free zones. Actually, all of the most recent European BT vaccination campaigns have used exclusively inactivated vaccines. The use of inactivated vaccines avoid risk associated with the use of live-attenuated vaccines, such as reversion to virulence, reassortment of genes with field strain, teratogenicity and insufficient attenuation leading to clinical disease. The mass vaccinations of all susceptible animals are the most efficient veterinary method to fight against BT and successful control of disease. The vaccination of livestock has had a major role in reducing BTV circulation and even in eradicating the virus from most areas of Europe.

Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus.

The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination.

Monitoring studies of bluetongue disease in ruminants imported to Poland from EU.

The results of 4-year serological and 2-year virological monitoring studies of bluetongue (BT) disease in ruminants imported to Poland from EU countries are presented. Sera were screened using the c-ELISA and direct ELISA tests. Out of 49587 samples of sea, 3385 (6.83% of a total number of samples) were found to be positive for bluetongue virus (BTV). The majority of seropositive animals were imported to Poland from Germany, Czech Republic and The Netherlands. The high number of positive animals detected in 2008-2009 was the result of bluetongue vaccination implemented in BTV-infected EU countries in 2008. The rRT-PCR was applied to detect viral RNA in blood samples. The presence of BTV RNA was found in 38 samples of blood collected from German cows, one sample from Dutch fallow deer and one sample from a 4-week-old calf born from BT positive dam imported from Germany. On the basis of present results it can be stated that the implemented virological surveillance investigations should be continued to monitor the actual BT status in Poland. As it is impossible to differentiate infected from vaccinated animals and deduce whether the animals have been or have not been vaccinated properly, the continuation of serological monitoring studies using actually available ELISA kits should be reconsidered.

Application of real-time reverse transcription polymerase chain reaction for the detection of SVDV.

Application of real-time RT-PCR (rRT-PCR) for detection of swine vesicular disease virus (SVDV) in samples of archival SVDV isolates and clinical samples collected from SVDV infected pigs was described. A primer set that targets the IRES region of the SVDV genome and TaqMan probe specific for a highly conserved region in SVDV RNA IRES region were used. The assay detected viral RNA in all tested archival strains of SVDV isolated in Europe during years 1972-73 and 1992 as well as in clinical samples collected from experimentally infected pigs. The rRT-PCR can provide quantitative and qualitative information and is more sensitive and faster to perform than the conventional RT-PCR.

Seroprevalence of antibodies against bluetongue virus in animals imported to Poland from EU countries.

The aim of this study was to determine the seroprevalence of BTV-specific antibodies in animals imported to Poland from EU countries after 15 June 2006. From 1 January 2007 to 22 January 2008, a total of 10719 samples of sera collected from cattle, goats and fallow deer were tested. Sera were screened using the highly sensitive and specific c-ELISA test and positive results were confirmed by the AGID assay. Out of 10719 sera, 30 (0.28% of the total number of samples) were found to be positive in both tests applied. All of 21 seropositive cattle specimens were imported to Poland from Germany whereas 9 seropositive fallow deer were of Dutch origin. In conclusion, it can be stated that because BTV situation in Europe is getting worse, implemented surveillance studies should be continued to monitor the actual BT status in Poland.

Detection of foot-and-mouth disease virus infection in vaccinated cattle.

The aim of this study was to evaluate the value of commercially available kits for the detection of foot-and-mouth disease (FMD) virus infection in vaccinated cattle. The cattle were vaccinated with a commercial aqueous FMD vaccine type A24 and subsequently challenged 28 days post vaccination with homologous FMD virus. Seven of eight animals were protected from clinical disease and all became carriers. They were bled sequentially for up to 130 days post infection and samples of sera were tested with three ELISA kits: CHEKIT FMD-3ABC, Ceditest FMDV-NS and SVANOIR FMDV 3ABC-Ab ELISA. The Ceditest kit appears to be relatively higher sensitive than the others. When examined with this ELISA, all cattle developed of FMDV nonstructural proteins (NSPs) antibodies and remained positive throughout the period of the experiment. The response of antibodies against 3ABC antigen delayed in two cattle challenged with FMDV A24 virus. One of the cattle reacted negatively in Svanoir ELISA kit and sera from two animals were found negative in CHEKIT ELISA. It can be concluded that all tested kits can be a promising tool for FMD control and eradication campaigns in situation where emergency vaccination was applied.

Prevalence of seroreagents to FMDV in the cattle population in Poland: results of 9-year monitoring studies.

The aim of this study was to evaluate the occurrence of antibodies to foot-and-mouth disease virus (FMDV) in sera of cattle in Poland. The examinations were performed using the virus neutralization (VN) test and ELISA methods: liquid-phase blocking ELISA (LPBE) and 3ABC-ELISA. During 1993-2001, about 681,000 samples of sera collected from animals held on the territory of Poland were tested. Of about 600,000 sera taken from animals exported to the European Union, 963 samples (0.16%) were found to be positive to FMDV types A, O and/or C. During 1996-2001 out of 85,000 sera tested as part of the national serological surveillance program for FMD, the FMDV antibodies were recorded in 51 (0.06%) samples. Persistence of FMD antibodies was observed in sera of cattle from the region around Zdunska Wola, which had been vaccinated annually during 1985-1985 with trivalent FMD vaccine. The results of the serological studies of 550 animals from this region indicates the presence of FMDV antibodies in sera of 240 (44%) cattle. A half-life of maternal antibodies in sera of calves born to seropositive dams was estimated; the highest level of FMDV antibodies was detected in sera taken from new-born calves aged 5-10 days. The level of FMDV antibodies in beestings of dams was highest during the first 10 hours after parturition; after 24 hours a significant decrease (3-5 times) was found and in two weeks post parturition FMDV antibodies were undetectable in the milk. It was established that all LPBE/VN positive sera of cattle exported from Poland, from the vaccination zone around Zdunska Wola as well as those tested as part of the national serological surveillance program for FMD, were taken exclusively from vaccinated animals or calves born to vaccinated dams.

Quantitation of foot-and-mouth disease virus genomes in bovine tissue by competitive RT-PCR.

The sensitivity of a reverse transcription-dependent polymerase chain reaction (RT-PCR) for detecting foot-and-mouth disease virus (FMDV) genomes was quantified by use of RNA transcribed in vitro from FMDV-specific cDNA. Previously, the cDNA had been elongated by 228 base pairs. The minimum number of template molecules required to obtain the specific RT-PCR product was determined to be 10(4). This was achieved by use of 1 microg of primer for cDNA synthesis and by undertaking of at least 30 cycles of PCR. Knowing the sensitivity of the system prompted the examination of clinical samples for content of FMDV genomes. The samples were tongue and foot epithelia as well as nasal discharge, removed 11-14 days after infection from 14 cattle. They all contained FMDV genomes but not in each clinical specimen. The size difference of the products amplified from transcript and viral genome enabled the estimate by competitive RT-PCR of the number of viral genomes contained in some samples. The RNA extracted from approximately 10(7) tissue cells each was found to contain between less than 10(6) and up to 10(8) FMDV genomes, irrespective of the sample type.

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.

Foot-and-mouth disease virus O1Lombardy is biochemically related to O2 isolates.

The capsid protein VP1-encoding RNA regions of the foot-and-mouth disease virus isolates O1Lombardy/1946 and O2Brescia/1947 were sequenced and found to be closely related to each other and to O2Normandy/1949, despite some sequence differences. The O1Lombardy sequence was expected to be more closely related to those of the subtype O1 isolates of 1965 and later (e.g., O1Kaufbeuren/1966), but this was not the case. The serological subtyping of both the Lombardy and the Kaufbeuren isolate as O1 strains was possibly due to identical VP1 C-terminal sequences, since all the subtype O2 isolates differ here from the O1 isolates at residue 209. Considerable dissimilarity of other O1Lombardy and O2Brescia genome parts to those of O1Kaufbeuren was qualitatively shown by analyzing the sizes of RNase-treated hybrids formed with virus RNA and defined subgenomic fragments of O1Kaufbeuren-specific antisense cRNA. These hybrids were fragmented into oligonucleotides, but others containing O1Kaufbeuren virus RNA were protected.

Effect of prolactin, progesterone, pregnancy and lactation on DNA synthesis and DNA polymerase activities in rabbit mammary gland.

DNA synthesis and DNA polymerase-alpha, -beta and -gamma activities in the rabbit mammary gland were studied during hormone-directed cellular growth. It was found that during pregnancy, early lactation and after injection of prolactin, changes in the activity of DNA polymerase-alpha paralleled the rate of mammary gland DNA synthesis. It was also found that the amount of polymerase-alpha activity bound to isolated chromatin depended on the physiological state of the animal. During pregnancy and early lactation changes in the activity of chromatin-bound enzyme correlated directly with the rate of DNA synthesis (r = 0.83). Moreover, in virgin rabbits treated with prolactin the activity of chromatin-bound DNA polymerase-alpha increased markedly at the same time as the DNA-synthetic rate increased. No correlation of the DNA-synthetic rate was found with the activity of soluble (cytosolic) DNA polymerase-alpha or with the activity of soluble or chromatin-bound DNA polymerases-beta and -gamma. On the basis of these results it is suggested that in the developing mammary gland both the activity and cellular distribution of DNA polymerase-alpha might be subject to hormonal regulation.

DNA polymerases of rabbit mammary gland: partial purification, characterization and changes in DNA polymerase activities as a function of physiological state.

DNA polymerases alpha and beta were partially purified from rabbit mammary gland, and their properties were examined. Many of these properties (sedimentation coefficients, pH optima, divalent cation requirements, sensitivity to various inhibitors) are similar to those commonly regarded as typical of eukaryotic DNA polymerases alpha and beta. Effect of stage of pregnancy and lactation on the levels of activity of DNA polymerases alpha, beta and gamma in rabbit mammary gland was examined. These experiments reveal a considerable variation of DNA polymerase-alpha activity; the highest enzyme activity is observed on day-20 of pregnancy and on day-1 of lactation.

Calcium and insulin in the regulation of DNA synthesis in mouse mammary gland studied in organ culture.

The interaction of calcium and insulin was investigated in mouse mammary gland organ culture by analysis of the time-course of the DNA-synthetic response following delayed addition of insulin or calcium to the culture media. The DNA-synthetic responses were also studied using mammary explants from virgin, pregnant and lactating mice. These experiments have shown that qualitative differences exist in the responsiveness of mammary epithelium to insulin and calcium. Thus, although it is evident that in mammary epithelium calcium modifies the DNA-synthetic response to insulin, the present data do not support the concept that Ca2+ is the intracellular mediator of insulin action.

Variation of DNA polymerase activities and DNA synthesis in mouse mammary gland during pregnancy and early lactation.

The rate of DNA synthesis and the activity of DNA polymerases and thymidine kinase were measured during the endocrine-regulated cellular growth and differentiation of mouse mammary gland. Using specific assays, the activity of the DNA polymerases, alpha, beta and gamma, was determined in tissue extracts of mammary glands of mice at various stages of pregnancy and early lactation. In addition, extracts of the mammary tissue of virgin, mid-pregnant and early lactating mice were fractionated on sucrose density gradients, and the activity of DNA polymerase alpha and beta was assayed in the gradient fractions. It was demonstrated that the activity of DNA polymerase alpha varied considerably during pregnancy and after parturition, showing peaks on day 12 of pregnancy and days 3-4 of lactation. In pregnancy, there was an apparently parallel correlation between the amount of DNA-polymerase-alpha activity and the rate at which the cells incorporated labelled thymidine into DNA, but the relationship was less clearly expressed during early lactation. The activity of the DNA polymerases, beta and gamma, as well as that of thymidine kinase showed little variation during these periods. Thus, in the developing mammary gland, no correlation was found between DNA synthesis and the activity of the DNA polymerases, beta and gamma, or thymidine kinase.

Characterization of DNA polymerases from the mammary gland of pregnant rabbit.

1. The nuclear extract from the mammary gland of pregnant rabbit was shown to contain DNA polymerases alpha, beta and gamma. Polymerases alpha and gamma were found also in the mitochondria-free cytoplasmic fraction. 2. The nuclear enzymes were partially purified, separated by sucrose-density-gradient centrifugation, and identified on the basis of their template-primer utilization, sedimentation coefficients and sensitivity to N-ethylmaleimide. 3. Catalytic properties of the activated-DNA-dependent DNA polymerases alpha and beta were also described.