Formulation, processing and water stability of pelleted feeds with varying levels of protein used in carp nutrition studies

Four dry pelleted feeds containing 20%, 30%, 40% and 45% protein were formulated incorporating casein as the main source of protein for use in carp nutrition studies. The caloric content in all the feeds was maintained constant. The method of processing is described. The formulated diets were tested for water stability. This test has revealed that the diet containing 20%, 30% and 40% protein had better stability than that containing 45% protein. This was due to the relatively higher fat content in the former three diets. However, all the feeds were sufficiently stable at the end of one hour in which time carps are known to utilise supplementary diets

Modelling the economic efficiency of using different strategies to control Porcine Reproductive & Respiratory Syndrome at herd level

PRRS is among the diseases with the highest economic impact in pig production worldwide. Different strategies have been developed and applied to combat PRRS at farm level. The broad variety of available intervention strategies makes it difficult to decide on the most cost-efficient strategy for a given farm situation, as it depends on many farm-individual factors like disease severity, prices or farm structure. Aim of this study was to create a simulation tool to estimate the cost-efficiency of different control strategies at individual farm level. Baseline is a model that estimates the costs of PRRS, based on changes in health and productivity, in a specific farm setting (e.g. farm type, herd size, type of batch farrowing). The model evaluates different intervention scenarios: depopulation/repopulation (D/R), close & roll-over (C&R), mass vaccination of sows (MS), mass vaccination of sows and vaccination of piglets (MS + piglets), improvements in internal biosecurity (BSM), and combinations of vaccinations with BSM. Data on improvement in health and productivity parameters for each intervention were obtained through literature review and from expert opinions. The economic efficiency of the different strategies was assessed over 5 years through investment appraisals: the resulting expected value (EV) indicated the most cost-effective strategy. Calculations were performed for 5 example scenarios with varying farm type (farrow-to-finish – breeding herd), disease severity (slightly – moderately – severely affected) and PRRSV detection (yes – no). The assumed herd size was 1000 sows with farm and price structure as commonly found in Germany. In a moderately affected (moderate deviations in health and productivity parameters from what could be expected in an average negative herd), unstable farrow-to-finish herd, the most cost-efficient strategies according to their median EV were C&R (€1′126′807) and MS + piglets (€ 1′114′649). In a slightly affected farrow-to-finish herd, no virus detected, the highest median EV was for MS + piglets (€ 721′745) and MS (€ 664′111). Results indicate that the expected benefits of interventions and the most efficient strategy depend on the individual farm situation, e.g. disease severity. The model provides new insights regarding the cost-efficiency of various PRRSV intervention strategies at farm level. It is a valuable tool for farmers and veterinarians to estimate expected economic consequences of an intervention for a specific farm setting and thus enables a better informed decision

Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses.

Recently, we identified a unique -2/-1 ribosomal frameshift mechanism in PRRSV, which yields two truncated forms of nonstructural protein (nsp) 2 variants, nsp2TF and nsp2N. Here, in vitro expression of individual PRRSV nsp2TF and nsp2N demonstrated their ability to suppress cellular innate immune responses in transfected cells. Two recombinant viruses were further analyzed, in which either nsp2TF was C-terminally truncated (vKO1) or expression of both nsp2TF and nsp2N was knocked out (vKO2). Host cellular mRNA profiling showed that a panel of cellular immune genes, in particular those involved in innate immunity, was upregulated in cells infected with vKO1 and vKO2. Compared to the wild-type virus, vKO1 and vKO2 expedited the IFN-α response and increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Our data strongly implicate nsp2TF/nsp2N in arteriviral immune evasion and demonstrate that nsp2TF/nsp2N-deficient PRRSV is less capable of counteracting host innate immune responses

A comparative evaluation of avidin-biotin ELISA and micro SNT for detection of antibodies to infectious bovine rhinotracheitis in cattle population of Odisha, India

Aim: The present study was undertaken to serologically detect Infectious Bovine Rhinotracheitis (IBR) in the cattle population of Odisha, India using micro-Serum neutralization test (micro SNT) and Avidin-Biotin Enzyme linked immuno sorbent assay (AB ELISA) and finding out their comparative efficacy to serve as a suitable diagnostic tool in field condition. Materials and Methods: The study was carried out using serum samples (n=180) collected randomly from cattle populations of nine districts of Odisha. Similarly vaginal swabs (n=26) from cattle having history of repeat breeding, abortion, vulvo-vaginitis and nasal swabs (n=8) from calves with respiratory symptoms and nasal discharge were collected aseptically, to ascertain the circulation of virus among the cattle population. Results: Virus isolation by cell culture and subsequent confirmation by polymerase chain reaction confirmed four isolates. Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively. The sensitivity and specificity of AB ELISA test was found to be 88.23% and 95.70% respectively taking micro SNT as gold standard and the kappa value between the two tests was 0.75. Conclusion: Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively, thus highlighting the circulation of virus among the livestock population of Odisha and that AB ELISA could be more efficiently applied for the sero-diagnosis of IBR virus infections at field conditions, with demand for more study on faster, efficient and large scale screening of the infected animals

Mutations in a Highly Conserved Motif of nsp1? Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus

Citation: Li Y, Shyu D-L, Shang P, Bai J, Ouyang K, Dhakal S, Hiremath J, Binjawadagi B, Renukaradhya GJ, Fang Y. 2016. Mutations in a highly conserved motif of nsp1? protein attenuate the innate immune suppression function of porcine reproductive and respiratory syndrome virus. J Virol 90:3584–3599. doi:10.1128/JVI.03069-15.Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1? (nsp1?) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique ?2/?1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif (123GKYLQRRLQ131) reduced the ability of nsp1? to suppress interferon beta (IFN-?) activation and also impaired nsp1?'s function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1? mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-? expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1? mutants, IFN-? production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-? in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1? function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals

Nanoparticle-based vaccine development and evaluation against viral infections in pigs

International audienceAbstractVirus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. The economic burden caused by virus infections such as Porcine Reproductive and Respiratory Syndrome Virus, Swine influenza virus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus 2, Foot and Mouth Disease Virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. Pigs can also have a role in zoonotic transmission of some viral infections to humans. Inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. Thus, improvements over existing vaccines are necessary to: (1) Increase the breadth of protection against evolving viral strains and subtypes; (2) Control of emerging and re-emerging viruses; (3) Eradicate viruses localized in different geographic areas; and (4) Differentiate infected from vaccinated animals to improve disease control programs. Nanoparticles (NPs) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. NPs help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. Some of the NPs-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (M) cells and dendritic cells (DCs). In conclusion, NPs-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. This review highlights the recent developments in NPs-based vaccines against porcine viral pathogens and how the NPs-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy

Development and validation of RP-HPLC method for the determination of Darifenacin Hydrobromide in bulk drug and pharmaceutical dosage form

The main objective of present study is to develop and validate a new, simple, precise and accurate RP-HPLC method for the determination of Darifenacin Hydrobromide (DFH) in bulk and pharmaceutical dosage forms. The separation and quantification of the drug was achieved on a RP C18 column (250×4.6mm, 5μm) using a mobile phase of acetonitrile: buffer (50:50), pH 3.0 ± 0.2 at a flow rate of 1 mL/min with detection of analyte at 287 nm. The separation was achieved with in 4.0 ± 0.3 min. The method showed good linearity in the range of 10-100 μg/mL. The intra and inter day RSD ranged from 0.20-0.58%. The recovery (mean ± SD) of low, medium and high concentrations were 98.50 ± 0.20, 100.27 ± 0.15 and 100.90 ± 0.09 respectively. The limit of detection and limit of quantification were 0.31 and 0.61 μg/mL, respectively. It can be concluded that the present method could be superior over the methods which were reported earlier.&nbsp

Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus

A recombinant baculovirus expressing membrane bound form of hemagglutinin-neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263-368 and 538-609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay

Smart contracts vulnerabilities detection using ensemble architecture of graphical attention model distillation and inference network

Smart contracts are automated agreements executed on a blockchain, offering reliability through their immutable and distributed nature. Yet, their unalterable deployment necessitates precise preemptive security checks, as vulnerabilities could lead to substantial financial damages henceforth testing for vulnerabilities is necessary prior to deployment. This paper presents the graphical attention model distillation and inference network (GAMDI-Net), a pioneering methodology that significantly enhances smart contract vulnerability detection. GAMDI-Net introduces a unique graphical learning module that employs attention mechanism networks to transform complex contract code into a smart graphical representation. In addition to this a dual-modality model distillation and mutual modality learning mechanism, GAMDI-Net excels in synthesizing semantic and control flow data to predict absent bytecode embeddings with high accuracy. This methodology not only improves the precision of vulnerability detection but also addresses scalability and efficiency challenges, reinforcing trust in the deployment of secure smart contracts within the blockchain ecosystem

Evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes chronic, economically devastating disease in pigs of all ages. Frequent mutations in the viral genome result in viruses with immune escape mutants. Irrespective of regular vaccination, control of PRRSV remains a challenge to swine farmers. In PRRSV-infected pigs, innate cytokine IFN-α is inhibited and the adaptive arm of the immunity is delayed. To elucidate both cellular and innate cytokine responses at very early stages of PRRSV infection, seven weeks old pigs maintained on a commercial pig farm were infected and analyzed.</p> <p>Results</p> <p>One pig in a pen containing 25 pigs was PRRSV infected and responses from this pig and one penmate were assessed two days later. All the infected and a few of the contact neighbor pigs were viremic. At day 2 post-infection, approximately 50% of viremic pigs had greater than 50% reduction in NK cell-mediated cytotoxicity, and nearly a 1-fold increase in IFN-α production was detected in blood of a few pigs. Enhanced secretion of IL-4 (in ~90%), IL-12 (in ~40%), and IL-10 (in ~20%) (but not IFN-γ) in PRRSV infected pigs was observed. In addition, reduced frequency of myeloid cells, CD4^-CD8⁺T cells, and CD4⁺CD8⁺T cells and upregulated frequency of lymphocytes bearing natural T regulatory cell phenotype were detected in viremic pigs. Interestingly, all viremic contact pigs also had comparable immune cell modulations.</p> <p>Conclusion</p> <p>Replicating PRRSV in both infected and contact pigs was found to be responsible for rapid modulation in NK cell-meditated cytotoxicity and alteration in the production of important immune cytokines. PRRSV-induced immunological changes observed simultaneously at both cellular and cytokine levels early post-infection appear to be responsible for the delay in generation of adaptive immunity. As the study was performed in pigs maintained under commercial environmental conditions, this study has practical implications in design of protective vaccines.</p