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Open access

Aneta Pluta, Marzena Rola-Łuszczak, Monika Olech and Jacek Kuźmak

Abstract

In this study the sequences of the long terminal repeat (LTR) of field isolates of the bovine leukaemia virus (BLV) were analysed. These isolates came from emerging cases of BLV infection in cattle from herds having BLV-free status. We found several sequence variations within regulatory motifs in the LTRs like GRE, DAS and interferon binding site. These mutations can possibly affect transcriptional activity of the virus, leading to its silencing.

Open access

Magdalena Materniak-Kornas, Zbigniew Osiński, Marcin Rudzki and Jacek Kuźmak

Abstract

Introduction: Infections with bovine foamy virus (BFV) were found in many countries but there is a lack of large-scale surveys on the prevalence of BFV among dairy cattle. The aim of this study was to develop and validate the recombinant Gag protein-based ELISA and to estimate the prevalence of antibodies against BFV. Material and Methods: Gag coding region from BFV was cloned into expression vector pT7Arg-STOP, which expressed a high level of recombinant Gag protein from E.coli. The ELISA was standardised, and the cut-off value and sensitivity and specificity of the test were calculated using a receiver operating characteristic and Bayesian estimation. Results: A total of 3,051 serum samples were tested by ELISA and 939 (30.8%) sera were recognised as positive. When Bayesian approach was used, the overall true BFV prevalence was 29.7% (95% CI: 25.9-33.4%). Conclusion: Expressed Gag protein of BFV has been used successfully as an antigen for ELISA. Eventually, this study provides basic information about the epidemiological status of infection with BFV in dairy cattle in Poland, which can be used for further studies on dissemination and transmission of BFV infection.

Open access

Marzena Rola-Łuszczak, Agnieszka Grabowska, Bogusław Szewczyk and Jacek Kuźmak

Abstract

Introduction: Field isolates of bovine leukaemia virus (BLV) show the presence of a few amino acid substitutions in major conformational G and H epitopes on surface glycoprotein gp51. Potentially, these substitutions can affect the 3D structure of these epitopes leading to their diminished immunoreactivity. The aim of this study was to express three gp51 glycoproteins carrying mutated epitopes as recombinant baculovirus proteins in insect cells to test their immunoreactivity with bovine sera.

Material and Methods: Env gene chimeras encoding mutated epitopes G and H in the env backbone of BLV FLK strain were constructed, cloned into pFastBac1 vector, and expressed in baculovirus.

Results: The presence of recombinant gp51 protein in Sf9 insect cells was confirmed using monoclonal antibodies. ELISA tests were developed to check the immunoreactivity of recombinant protein with bovine sera.

Conclusion: Recombinant gp51 proteins with altered G and H epitopes can be used for further studies to analyse the serological response of bovine sera towards BLV antigenic variants.

Open access

Ewelina Iwan, Maria Szczotka and Jacek Kuźmak

Abstract

The aim of the study was to develop an in situ PCR (IS-PCR) method for detection of bovine leukemia virus (BLV) in cell cultures. Samples from five BLV positive and five BLV negative cows were collected and dendritic cells (DCs) from blood, bone marrow, spleen, and lymph node were cultured. Cultures prepared from healthy animals were infected with BLV. After two weeks, the cells were tested by nested PCR and IS-PCR for the presence of proviral DNA. As a positive control adherent cell line permanently infected with BLV was used. BLV was successfully detected by IS-PCR in DCs from naturally infected cattle and DCs infected in vitro. In control, non-infected DCs, the results of the reaction were negative. The results of provirus detection by IS-PCR were similar with these performed with nested PCR. Additionally, IS-PCR provides many advantages, like specific localisation of infection and smaller number of cells needed as template for PCR.

Open access

Monika Olech, Marzena Rola-Łuszczak, Bożenna Kozaczyńska, Piotr Kubiś, Aneta Pluta, Anna Gil and Jacek Kuźmak

Abstract

In the study, a 122 bp fragment of gag gene encoding immunodominant epitope on capsid protein of small ruminant lentiviruses (SRLVs) found in sheep was amplified by PCR and analysed by SSCP and sequencing. Out of 30 DNA samples, five showed different migration patterns, demonstrating the individual variations within gag sequences, which were confirmed afterwards by sequence analysis. In two samples nucleotide changes yielded amino acid substitutions highlighting the conservative nature of gag encoded immunoreactive epitope but also potencial insensitivity of a single-strain-based immunoassay.

Open access

Monika Olech, Piotr Kubiś, Czesława Lipecka, Andrzej Junkuszew, Tomasz M. Gruszecki and Jacek Kuźmak

Abstract

The aim of this study was to investigate the presence of proviral DNA and colostral antibodies in lambs born to and fed by ewes infected with small ruminant lentiviruses (SRLV). It was demonstrated that all 20 lambs tested 24 h after colostrum ingestion were serologically positive with high antibody titres. These gradually decreased with time, and at week 12 all lambs were seronegative. Twenty percent of lambs tested at the 2nd week postpartum were provirus positive by qPCR as a result of consumption of infected colostrum or in utero infection. When tested at three months of life, 95% of the lambs were provirus positive, probably as a result of horizontal transmission of the virus. Since these animals could play an important role in the early propagation of SRLV to susceptible herdmates, early removal of provirus-positive animals could help to prevent new infections.

Open access

Czesława Lipecka, Andrej Junkuszew, Jacek Kuźmak, Tomasz M. Gruszeck, Bożena Kozaczyńska, Monika Olech, Wiktor Bojar and Zbigniew Osiński

Abstract

The study included a sheep flock comprising five genetic groups. The ELISA was applied to perform constant monitoring (every six months) for the infection of ewes with small ruminant lentivirus (SRLV). The research results demonstrated a negative effect of SRLVs infection on lamb rearing that, depending on the genetic group, proved to be lower 1.3%-1.4% compared to the seronegative mothers. At relatively equal fertility (94%-100%) and more differentiated prolificacy (179%-198%) in all the examined groups (except the Suffolk breed), a rearing index was higher in the seronegative animals 6.8%-24.1% compared to the seropositive mothers. The Suffolk breed proved to be the genetic group most susceptible to SRLV infection. A prolificacy of infected ewes was 10% lower, a lamb rearing rate was 13% lower , and a general reproductive performance was 18% lower in comparison to healthy ewes.