Telomeres are repetitive sequence structures at the ends of chromosomes. They consist of the double stranded DNA repeats followed by the short single stranded DNA. In humans and other verterbrates the telomeric sequence is composed of tandem of TTAGGG repeats. With each cells division telomeres shorten by up to 200 base pairs. Telomerase is an enzyme responsible for continuous cell growth and is repressed in most somatic cells, except proliferating progenitor cells, but in more than 85% of cancer cells telomerase expression is observed. Tumour cells with metastatic potential may demonstrate a high telomerase activity, allowing cells to escape from the inhibition of cell proliferation due to shortened telomeres. Determination of telomerase expres- sion was performed with the use of PCR ELISA in samples isolated from bovine leukaemia virus (BLV) infected cows. Telomerase activity was found in almost all investigated samples. The relative telomerase activity (RTA) was higher in infected cows than in healthy animals and the differences were statistically significant (α=0.05). In blood lymphocytes of BLV-infected cows the mean values of telomerase expression determined in real-time PCR were 3534.12 copies, in the healthy group there were 1010.10 copies and these differences were also statistically significant. For telomere length evaluation the Telomere PNA/FITC FISH and Telomere PNA/FITC FISH for flow cytometry were used. The mean fluorescence intensity of telomere sequences calculated on the surface of interphase nuclei of leukaemic blood lymphocytes was lower than that in the control animals and the difference was statistically significant. The mean length of telomeres in BLV- infected and healthy cows was 31.63 ± 12.62 and 38.4 ± 4.03, (p=0.112), respectively.

Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/μL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demon- strated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/μL for BPV and BCoV, and 2.0×101 copies/μL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.

Herpesviruses (HV) are pathogens causing infections in humans and animals worldwide. Since it shares many common features with other HV, bovine HV type 1 (BoHV-1) was selected as a model to test the anti-herpesviral activity of medicinal plants.

Fifteen plants were chosen in this study for their medical, antibacterial and antiviral properties. The aim was to investigate ethanolic extracts from the selected medicinal plants for anti-BoHV-1 activity. The virucidal activities were evaluated by comparing the effect of noncytotoxic concentrations of extracts on BoHV-1 strain 1640 replication in Madin-Darby bovine kidney (MDBK) cells. Virucidal activity was determined by means of virus titration after exposure to the extracts. The extract of Desmodium canadense was found to be the most effective virucide – the 50% tissue culture infective dose (TCID50) after exposure was 3.75 log10 and the virus reduction factor was ≥5.0±0.25 log10. The extract of D. canadense was therefore chosen for further studies. Virus yield reduction assays showed that D. canadense extract had time-dependent and dose-dependent effects. It effectively reduced virus titre from 8.33 log10 to 4.67 log10 (p<0.01). The virucidal activity was also confirmed by real-time polymerase chain reaction (real-time PCR), where the number of threshold cycles (Ct) was inversely proportional to the virus titre in TCID50 The virucidal activity was also confirmed by real-time polymerase chain reaction (real-time PCR). This method showed that the number of threshold cycles (Ct) was inversely proportional to the virus titre (direct correlation with exposure time R=0.9321). The extract of D. canadense showed a high virus reduction capacity. In future, such active substances should be identified for the development of effective antivirals.

Aim: The aim of this study was to analyze the effect of bovine follicular fluid on the survival, morphology and kinetic parameters of bovine thawed spermatozoa under laboratory conditions.

Materials and methods: The semen from 5 bulls of proven fertility was incubated in follicular and physiological fluid for 8 hours. During this time assessment using the CASA system was performed. At the beginning and the end of incubation process evaluation by flow cytometry was conducted.

Results: The results of the sperm motility assessment showed a significant decrease in the analyzed parameters both in the follicular and physiological fluid. A significant reduction in all parameters characterizing movement properties in the semen incubated in the follicular fluid was found. In the physiological fluid, a similar trend was demonstrated only for the following proper- ties: VAP, VSL, VCL, ALH, BCF. A significant difference was found for both fluids in: VCL (p=0.026), ALH (p=0.038) and LIN (p<0.001) at the beginning of incubation. The results of the plasma membrane integrity assessment showed a statistically significant increase in the percent- age of dying sperm at the 8th hour of the incubation in the follicular fluid. In the case of semen incubation in physiological fluid, a statistically significant decrease in the percentage of live non-damaged cells was found with a simultaneous increase in the subpopulation of undamaged dead cells.

Conclusions: Follicular fluid rapidly accelerates the capacitation process. The results of flow cytometry support the hypothesis concerning the ability of follicular fluid to prolong sperm sur- vival.

Dendritic cells (DCs) due to their ability to present antigens are essential during the immune response to infections. The aim of the study was to evaluate the influence of bovine leukaemia virus (BLV) infection on DC properties. Cytokine profiles of myeloid, plasmacytoid and mono- cyte derived DCs from BLV infected cattle were analysed. Concentrations of IL-6, IL-10, IL-12, IFN-γ, and TNF-α in DC cultures were measured by flow cytometry. Obtained results indicated activation of pDCs population, where a significant increase in production of the IFN-γ was shown. Meanwhile, a decrease in production of IFN-γ and increase in production of IL-10 were shown in mDCs; the main population responsible for antigens presentation. This may indicate a contribu- tory role of the population during the process of persistent infection. In MoDCs population a significant elevation in secretion of proinflammatory cytokines – IL-6 and TNF-α was noted.

Early recognition of altered lactate levels is considered a useful prognostic indicator in dis- ease detection for both human beings and animals. It is reasonable therefore to hypothesize that a portable, point of care (POC) spectrophotometric device for analysis of lactate levels, may have an application for field veterinarians across a range of conditions and diagnostic procedures. In this study, a total of 72 cattle in the transition period underwent POC spectrophotometric lactate measurement with a portable device (The Vet Photometer) in the field, with a small portion of blood used for comparative ELISA evaluation. Lactate measurements were compared using a of Passing-Bablok regression analysis and Bland-Altman plots. The Vet Photometer lactate mea- surement results were in agreement with those generated by the ELISA method. Values for the agreement were derived, in a 95% CI between -1.3 and 0.99, and a positive correlation (r=0.71) between the two measurements. The equation y= 0.68x + 0.60 was achieved using a Pass- ing-Bablok regression analysis. There were no statistical differences in mean values between the measurement methods. In conclusion, a novel veterinary POC spectrophotometric device “Vet Photometer” is an accurate device for evaluation of lactate levels in healthy transition cows.

Background: Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe.

Objective: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers.

Results: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomavirus 4 isolate 09 asi UK (Acc. No. MF384289) and 99% nucleotide identity with BPV-1 isolate EqSarc1 (Acc. No. JX678969). A comparison with BPV-1 isolate EqSarc1 revealed one silent mutation in C5827T which did not change the aminoacid codon. The remaining 6 isolates (28.6%) shared 100% nucleotide identity with the BPV-1 (Acc. No. X02346) “wild type” isolate, and 1 isolate (4.8%) demonstrated 99% nucleotide identity with BPV-2 (Acc. No. M20219).

Conclusions: Variants of BPV-1 isolate EqSarc1 (Acc. No. JX678969) constitute the most prevalent type of BPV-1 in Polish horses.

Injection of lymphokine activated killer (LAK) cells is known as useful for activation of cellular immune system. Although the effect of LAK cells has been clarified in human or mice, this effect on function of immune cells has not been examined in calves. Healthy ten Holstein calves were injected with the LAK cells 2 days after birth (LAK Group), and another eight calves were observed as controls (Control Group). All calves received the colostrum formulation on the day of birth, and then, were inoculated with a live attenuated vaccine of bovine herpesvirus (BHV)-1 at 2 (the first vaccination) and 6 (the second vaccination) weeks after birth. Peripheral blood of their dam obtained 3 weeks before calving was used for preparation of LAK cells. Blood samples were taken prior to vaccine inoculation and 3 days after the first inoculation, as well as 3 and 6 days after the second vaccination from all calves. Numbers of CD8+ and CD21+ cells increased significantly after the second vaccination in the LAK Group compared with Control Group. The present study suggested the improved effect of injecting LAK cells originated from dams on immune cells function of young calves after BHV-1 live vaccine.

In the present study on Bubalus bubalis of the Campania Region (Italy) the serum levels of derivatives of reactive oxygen metabolites (d-ROMs), anti-ROM and oxidative stress index (Osi) were evaluated. These data were then related to the seropositive status of the animals against alpha-herpesviruses, precisely Bubaline herpesvirus 1 (BuHV-1) and Bovine herpesvirus 1 (BoHV-1). Clinically healthy Mediterranean buffaloes were selected for this study. The serum samples of these animals were taken, and d-ROMs, anti-ROM and Osi were measured using commercially available tests. The preliminary data demonstrated that animals seropositive to both BuHV-1 and BoHV-1 present more oxidative stress than seronegative animals, as revealed by a significant increase in d-ROMs. Our results provide, for the first time, insight into the reac- tive oxygen species (ROS) modulation induced by the herpesvirus in Bubalus bubalis.

The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.