Passive radio frequency identification and video tracking for the determination of location and movement of broilers.

Phenotypes on individual animals are required for breeding programs to be able to select for traits. However, phenotyping individual animals can be difficult and time-consuming, especially for traits related to health, welfare, and performance. Individual broiler behavior could serve as a proxy for these traits when recorded automatically and reliably on many animals. Sensors could record individual broiler behavior, yet different sensors can differ in their assessment. In this study a comparison was made between a passive radio frequency identification (RFID) system (grid of antennas underneath the pen) and video tracking for the determination of location and movement of 3 color-marked broilers at d 18. Furthermore, a systems comparison of derived behavioral metrics such as space usage, locomotion activity and apparent feeding and drinking behavior was made. Color-marked broilers simplified the computer vision task for YOLOv5 to detect, track, and identify the animals. Animal locations derived from the RFID-system and based on video were largely in agreement. Most location differences (77.5%) were within the mean radius of the antennas' enclosing circle (≤128 px, 28.15 cm), and 95.3% of the differences were within a one antenna difference (≤256 px, 56.30 cm). Animal movement was not always registered by the RFID-system whereas video was sensitive to detection noise and the animal's behavior (e.g., pecking). The method used to determine location and the systems' sensitivities to movement led to differences in behavioral metrics. Behavioral metrics derived from video are likely more accurate than RFID-system derived behavioral metrics. However, at present, only the RFID-system can provide individual identification for non-color marked broilers. A combination of verifiable and detailed video with the unique identification of RFID could make it possible to identify, describe, and quantify a wide range of individual broiler behaviors.

Presence of Coxiella burnetii in dairy cattle and farms in the Czech Republic.

The aims of this study were to evaluate the prevalence of Coxiella burnetii on both herd and animal level based on ELISA and PCR tests. Antibodies to C. burnetii were detected in 22 out of the 24 bulk tank milk samples (91.6%) tested by ELISA and the IS1111 element of C. burnetii was detected in 10 out of the 24 samples (41.6%) by real-time polymerase chain reaction (PCR). ELISA testing showed individual seropositivity in 67 out of the 165 cows (40.6%) examined in 24 dairy cattle farms in different parts of the Czech Republic. Our study revealed that the prevalence of C. burnetii has increased substantially in the Czech Republic over the past 30 years, and that the causative agent is a potential risk factor for some reproductive problems in dairy farms and a possible risk factor for human infection.

Bayesian estimation of the true prevalence of paratuberculosis in Hungarian dairy cattle herds.

Paratuberculosis is a chronic incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), which leads to extensive economic losses on dairy farms, and may also pose serious public health risk to the consumers. The aim of our study was to estimate the true prevalence of paratuberculosis in commercial dairy cattle herds participating in a voluntary MAP testing programme that started in February 2018 in Hungary. Milk samples collected during official milk recording were used for MAP ELISA testing. A Bayesian two-stage hierarchical (herd and animal level) model was fitted to the data. Altogether, 26,437 cows from 51 herds were sampled, which represents 14.4 % of the Hungarian dairy cow population. The median herd size was 477 cows (interquartile range: 331-709). Each studied farm had at least one ELISA positive cow, resulting in a herd-level apparent prevalence of 100 %. The overall within herd apparent prevalence was 5.5 %. Herd-level true prevalence was estimated at 89.1 % [95 % credible interval (CrI): 80.3-95.6%]. Within the infected herds, the median animal-level true prevalence was 4.4 % (3.2-5.8%) for primiparous and 10.3 % (7.9-12.9%) for multiparous cows, respectively. The probability of having an animal-level true prevalence of at least 5% among primiparous cows, within infected herds, was 17.8 %. Similarly, the probability of having an animal-level true prevalence of at least 5% or 10 % among multiparous cows was 100 % and 56 %, respectively. Simulations assuming herd-level true prevalence varying from 50 to 100 % revealed high accuracy of our Bayesian model. Our study showed that a large percentage of the studied Hungarian dairy cattle herds was infected with MAP.

Lister strain of vaccinia virus armed with endostatin-angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer.

Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer.

Management practices associated with reproductive performance in Holstein cows on large commercial dairy farms.

As a result of the increase in herd size and the intensification of production, the complexity of reproductive management has been growing in dairy herds. The aim of our study was to examine the associations of management practices and reproductive performance in Holstein cows on large commercial dairy farms. Management practices applied to cows were surveyed between 22 May and 6 November 2015 in 34 large Holstein-Friesian dairy herds in Hungary. Individual data of 23 784 cows that calved between 1 January 2014 and 31 December 2014 in the surveyed herds were gathered. Associations between the management practices and the reproductive parameters were analyzed by mixed effects models. Regarding heat abatement we found that ventilation with sprinklers was associated with the shortest breeding interval (P0.05) results to lack of heat stress protection. It was also revealed, that lack of a well-established voluntary waiting period (VWP) or a VWP shorter than 50 days was associated with less days to first service (P<0.01), shorter breeding interval (P<0.01) and calving to conception interval (P<0.05), as well as higher odds of carrying a calf by 200 days in milk (P<0.01) compared with those using a VWP of at least 50 days. Those farms that applied transrectal ultrasonography were more likely to use ventilation with sprinklers (P<0.05), hormonal synchronization (P<0.01) and to perform early pregnancy diagnosis followed by pregnancy recheck (P<0.05). The application of transrectal ultrasonography with one of the aforementioned practices was associated with reduced days to first service (P<0.05), shorter breeding interval (P<0.05) and higher odds of pregnancy at 200 days in milk (P<0.05). Our study highlights the management practices most closely related to improved reproductive performance, which are, therefore, suggested to be applied on dairy farms, considering the local circumstances of the individual farms.

Associations between management practices and major reproductive parameters of Holstein-Friesian replacement heifers.

The aim of this study was to assess the relationship between the reproductive management practices and the performance of replacement heifers on large commercial dairy farms. The individual data of 14,763 heifers, first inseminated in 2014, were analysed from 33 Holstein-Friesian dairy herds in Hungary. The relationships between management practices and major reproductive parameters (age at first service, AFS; age at first calving, AFC; conception risk to first insemination, CR1; and pregnancy status at 20 months of age) were examined by mixed-effects models, with the herd as the random effect. The results showed that farms using oestrus detection aids experienced reduced AFS (p<0.001) and AFC (p=0.001). Observation of oestrus for shorter periods instead of continuously showed a tendency towards lower AFC (p=0.057) and was associated with higher odds of pregnancy at 20 months of age (p=0.020). Heifers on farms using sexed semen had younger AFS, but poorer CR1, compared to those using conventional semen exclusively (p<0.05). In addition, the odds of heifers being pregnant by 20 months of age was higher on farms with more experience using sexed semen (p=0.020). Frequent pregnancy diagnosis (i.e. more than once per week) was associated with younger AFC (p=0.023). Our results suggest the use of certain advanced reproductive management practices for heifer reproductive management in large dairy herds (e.g. oestrus detection aids), which can improve reproductive efficiency considerably, but are currently used only to a limited extent.

Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature.

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes.

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.

Large and small subunits of the Aujeszky's disease virus ribonucleotide reductase: nucleotide sequence and putative structure.

We determined the entire DNA sequence of two adjacent open reading frames of Aujeszky's disease virus encoding ribonucleotide reductase genes with the intergenic sequence of 9 bp. From the sequence analysis we deduce that ORFs encode large and small subunits, with sizes of 835 and 303 amino acids, respectively. Amino acid sequence comparison of ADV RR2 with that of equine herpesvirus type 1, bovine herpesvirus type 1, HSV-1 and varicella zoster virus revealed that 48% of amino acids represent clusters of residues conserved in all compared sequences. In the N-terminal part ADV RR1 shows low homology to the RR1 of other herpesviruses. Rest of the RR1 protein contains highly conserved amino acid sequences divided by blocks of low homology.

The distribution and aggregatability of intramembrane particles in phenylhydrazine-treated human erythrocytes.

Freeze-fracture analysis of phenylhydrazine-treated, unfixed human erythrocytes showed a random distribution of intramembrane particles both over membrane-bound Heinz-bodies and in the intervening areas when examined after fast freezing in liquid propane. The same results was obtained when unfixed, glycerinated red cells were frozen in liquid Freon. In contrast to previously published data (Low et al. (1985) Science 227, 531-533) these results indicate that binding of Heinz-bodies to the red cell membrane cannot cause morphologically detectable clustering of Band 3 in phenylhydrazine-treated red cells. Over numerous Heinz-bodies a decreased Acridine orange-induced particle aggregation was observed. The phenomenon of the oxidant-induced red cell fluorescence is described.

A comparative study on the cellular processing of free and gold-conjugated transferrin.

The kinetics of the transferrin-reticulocyte interaction was studied using free and colloidal gold-conjugated double labelled transferrin (Tf and AuTf, respectively). Simultaneous biochemical and morphological experiments provided the following information: 1. The cellular recycling of Tf is significantly faster than that of AuTf. 2. AuTf induces a marked increase in the number of multivesicular elements (MVE) in rabbit reticulocytes. 3. The release of AuTf from the cells is very slow and accumulation of gold particles in MVEs can be observed during the process. The results suggest that the two postulated pathways of the transferrin-cell cycle (a fast, iron-donating and a slow, receptor-shedding cycle) are not similarly involved in the cellular processing of Tf and AuTf. While it has been suggested that in the Tf-cell interaction the fast recycling mechanism is dominating, it is likely that, probably due to steric effects, the majority of AuTfs are forced into the slower receptor-shedding pathway. These observations call attention to the possible limitations of the colloidal gold labelling technique in the determination of the kinetics and pathway of intracellular processing of free ligands.

Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells.

Previous studies have shown that at least three polypeptides of 43, 39 and 38 kDa are translated from separate AUG codons of the thymidine kinase (TK) encoding mRNA of herpes simplex virus type 1. In addition, small tk-specific transcripts initiated within the tk coding region were observed. However, functional activity of these three proteins and their role in establishing of the TK+ cell phenotype is not yet clear. In order to locate the 5' boundary of the gene encoding functionally active TK, we constructed a set of deletion mutants with truncated 5' ends and examined their ability to provide a TK+ phenotype after microinjection into nuclei of LTK- cells. The results demonstrate that nucleotide sequences upstream from the second ATG codon can be removed without affecting the TK+ phenotype. Deletion of the second start codon and its downstream region inactivates the TK function. Those deletion mutants which contain only the third ATG codon are TK-. Thus, the 38-kDa polypeptide that initiates at the third start codon is not endowed with the TK+ activity. Constructs containing deletions up to nt +210 and lacking all 5'-end canonical and aberrant transcription control regions, as well as first start codon, can provide the TK+ function.

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5.

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRI, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been reduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA. Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.

Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells.

Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system. It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site. Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene. beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons. Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides. Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.

Lambda phagemids and their transducing properties.

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.

The catabolite gene activation system of E. coli may be directly involved in regulation of bacteriophage lambda development.

The primary structure of bacteriophage lambda DNA has been searched for the presence of consensus CAP binding sites. Four putative CAP binding sites have been found on the lambda genome, indicating that the catabolite gene activation system of E. coli may be directly involved in the regulation of lambda development. Molecular mechanisms of putative cAMP-CAP-mediated stimulation of lysogenic and lytic responses are discussed.

Replication and virulence of early protein 0 and long latency transcript deficient mutants of the Aujeszky's disease (pseudorabies) virus.

Early protein 0 (EP0)-deficient recombinant Aujeszky's disease viruses, Ka-ep0lac and Ba-ep0lac derived from strains Kaplan and Bartha, respectively, were constructed to explore the impact of the mutation on replication, virulence and latency of the virus. Inactivation of the EP0 gene resulted in a mutation of long latency transcript (Cheung et al., 1991) that is located on the complementary DNA strand of EP0 and immediate early protein (IE)175 genes. In infection of immortalized porcine kidney cells, the growth rate and yield of both EP0(-) mutant strains were significantly smaller than that of wild-type virus. Ka-ep0lac was found to be highly virulent, while Ba-ep0lac showed an attenuated phenotype in mice. PCR assay and immunohistochemistry showed that the Ba-ep0lac virus was able to establish latency in the mouse trigeminal ganglia. However, latent virus was not able to reactivate in explant reactivation assays. Accordingly, latent Ba-ep0lac has the potential to be exploited as vectors for the delivery of foreign genes to the nervous system.

Evaluation of combined vaccinia virus-mediated antitumor gene therapy with p53, IL-2, and IL-12 in a glioma model.

Our previous studies have shown that vaccinia virus (VV) expressing p53, interleukin-2 (IL-2), and interleukin-12 (IL-12) results in an effective inhibition of subcutaneous glioma growth in mice. We propose that combination therapy of tumors with virus-mediated p53 and cytokine genes offers the prospect of synergistic antitumor response. In this work, the antitumor efficacy of VV-mediated combination of p53, IL-2, and IL-12 genes was evaluated in a nude mouse model. To minimize cytokine-associated toxicity, a virus dose as low as 10 plaque-forming units of VV expressing IL-2 and IL-12 per animal was used alone and together with 2 x 10(7) plaque-forming units of VV expressing p53. Intratumoral treatment of established C6 glioma with recombinant viruses rVV-p53, rVV-mIL2, rVV-mIL12, and rVV-2-12 induced the prolonged expression of p53, IL-2, IL-12, and both cytokines simultaneously. The combination of rVV-p53/rVV-mIL 2 or rVV-p53/rVV-2-12 resulted in significant tumor inhibition compared to single modality treatment (P<.05). rVV-p53/rVV-2-12 therapy was associated with significant elevation of natural killer, Mac-1+, and NKT cells in blood and interferon-gamma, and tumor necrosis factor-alpha expression in tumors. The difference in the inhibition of tumor growth between the rVV-p53/rVV-mIL2 combination and rVV-p53 was statistically insignificant. These data demonstrate that gene therapy based on VV-mediated combination of p53, IL-2, and IL-12 treatment may be a promising adjunctive strategy for glioma treatment.

Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper.

Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy. The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses. Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses. In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection. In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome. In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV. The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E. coli beta-galactosidase. An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation.

Immunological characterisation of glycoprotein E of Aujeszky's disease virus.

A panel of 14 monoclonal antibodies (MAbs) against glycoprotein E (gE) of Aujeszky's disease (pseudorabies) virus (ADV), which constitutes a representative sample of naturally occurring gE-specific antibodies in sera from infected animals, was produced and characterised. Eleven topologically distinct antigenic domains represented by one or more MAbs were identified on gE by using these MAbs and three additional gE-specific MAbs. Three of the MAbs available recognised conformation-independent epitopes on gE, while the other 14 MAbs bound to conformation-dependent epitopes. By using the recombinant protein encompassing the N-terminal part of gE, which was expressed in Escherichia coli, all the conformation-independent epitopes of gE were mapped within the first 125 amino-terminal amino acids of gE. The epitopes of gE were demonstrated to be conserved among gE-positive laboratory, field and vaccine ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gE in naturally infected swine and immunised mice. Most of the infected animals responded weakly to the identified conformation-independent epitopes of gE, while the group of immunodominant epitopes of gE was represented exclusively by conformation-dependent antigenic determinants from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes play a crucial role in inducing the humoral immune response to gE of ADV during the natural infection of swine and immunisation of mice. The application of MAbs of our panel as research and diagnostic tools is discussed.