Pathway analysis of differentially expressed genes in Mycobacterium bovis challenged bovine macrophages.

The immune signalling genes during the challenge of bovine macrophages with bacterial products derived from tuberculosis causing bacteria in cattle were investigated in the present study. An in-vitro cell culture model of bovine monocyte-derived macrophages were challenged to Mycobacterium bovis. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.

Homologous prime-boost immunization with live attenuated Salmonella enterica serovar Senftenberg and its preventive efficacy against experimental challenge with various strains of S. Senftenberg.

BACKGROUND: The heterogeneity observed regarding persistence, and subsequent fecal shedding pattern of the Salmonella Senftenberg (S. Senftenberg) serovar in chicken's calls for development of the optimized immunization strategy which can provide protection against various S. Senftenberg isolated. Optimization of an immunization strategy with a live attenuated S. Senftenberg (Δlon and ΔcpxR) vaccine candidate (JOL1587) was undertaken in this study to evaluate the ability of a homologous prime-boost immunization strategy (using JOL1587) to confer protection against four different S. Senftenberg isolates in chickens. RESULTS: After oral immunization with JOL1587, the humoral, mucosal and cell-mediated immune responses were significantly higher in double immunized chickens than in single immunized and control group chickens. A significant increase in the multifunctional cytokine IL-6 and in helper and cytotoxic T cell populations after a booster immunization also indicated the advantage of double over single immunization. The four different S. Senftenberg field isolates were characterized by their persistence levels in chickens, and were subsequently used for challenge experiments to evaluate the differences in protective efficacy conferred by single and double immunization. Chickens from the doubleimmunized group exhibited significant reduction in the shedding of all four wild-type S. Senftenberg challenge strains below the detection limit in the fecal samples. Single immunized chickens showed a decrease in fecal shedding, but failed to exhibit complete protection against all the challenge strains. CONCLUSION: Although single immunization with JOL1587 showed a reduction in the fecal shedding of challenge strains, only the homologous prime-boost immunization strategy provided an adequate immune response for increased protection against all four challenge strains of S. Senftenberg from the feces of chickens.

Characterization and Evaluation of a Salmonella enterica Serotype Senftenberg Mutant Created by Deletion of Virulence-Related Genes for Use as a Live Attenuated Vaccine.

Natural infections of chickens with Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) are characterized by low-level intestinal invasiveness and insignificant production of antibodies. In this study, we investigated the potential effects of lon and cpxR gene deletions on the invasiveness of S Senftenberg into the intestinal epithelium of chickens and its ability to induce an immune response, conferring protection against S Senftenberg infection. With the allelic exchange method, we developed JOL1596 (Δlon), JOL1571 (ΔcpxR), and JOL1587 (Δlon ΔcpxR) deletion mutants from wild-type S Senftenberg. Deletion of the lon gene from S Senftenberg produced increased frequency of elongated cells, with significantly greater amounts of exopolysaccharide (EPS) than in the cpxR-deleted strain and the wild-type strain. The in vivo intestinal loop invasion assay showed a significant increase in epithelial invasiveness for JOL1596 (Δlon) and JOL1587 (Δlon ΔcpxR), compared to JOL1571 (ΔcpxR) and the wild-type strain. Furthermore, the S Senftenberg wild-type and mutant strains were internalized at high levels inside activated abdominal macrophages from chicken. The in vivo inoculation of JOL1587 (Δlon ΔcpxR) into chickens led to colonization of the liver, spleen, and cecum for a short time. Chickens inoculated with JOL1587 (Δlon ΔcpxR) showed significant increases in humoral, mucosal, and cellular immune responses specific to S Senftenberg antigens. Postchallenge, compared to the control group, the JOL1587 (Δlon ΔcpxR)-inoculated chickens showed not only lower persistence but also faster clearance of wild-type S Senftenberg from the cecum. We conclude that the increased intestinal invasiveness and colonization of internal organs exhibited by JOL1587 (Δlon ΔcpxR) led to the establishment of immunogenicity and conferred protective efficacy against S Senftenberg infections in chickens.

Duck gut viral metagenome analysis captures snapshot of viral diversity.

BACKGROUND: Ducks (Anas platyrhynchos) an economically important waterfowl for meat, eggs and feathers; is also a natural reservoir for influenza A viruses. The emergence of novel viruses is attributed to the status of co-existence of multiple types and subtypes of viruses in the reservoir hosts. For effective prediction of future viral epidemic or pandemic an in-depth understanding of the virome status in the key reservoir species is highly essential. METHODS: To obtain an unbiased measure of viral diversity in the enteric tract of ducks by viral metagenomic approach, we deep sequenced the viral nucleic acid extracted from cloacal swabs collected from the flock of 23 ducks which shared the water bodies with wild migratory birds. RESULT: In total 7,455,180 reads with average length of 146 bases were generated of which 7,354,300 reads were de novo assembled into 24,945 contigs with an average length of 220 bases and the remaining 100,880 reads were singletons. The duck virome were identified by sequence similarity comparisons of contigs and singletons (BLASTx E score, <10(-3)) against viral reference database. Numerous duck virome sequences were homologous to the animal virus of the Papillomaviridae family; and phages of the Caudovirales, Inoviridae, Tectiviridae, Microviridae families and unclassified phages. Further, several duck virome sequences had homologous with the insect viruses of the Poxviridae, Alphatetraviridae, Baculoviridae, Densovirinae, Iflaviridae and Dicistroviridae families; and plant viruses of the Secoviridae, Virgaviridae, Tombusviridae and Partitiviridae families, which reflects the diet and habitation of ducks. CONCLUSION: This study increases our understanding of the viral diversity and expands the knowledge about the spectrum of viruses harboured in the enteric tract of ducks.

Inhibition of Salmonella-induced apoptosis as a marker of the protective efficacy of virulence gene-deleted live attenuated vaccine.

Vaccination is one of the best protection strategies against Salmonella infection in humans and chickens. Salmonella bacteria must induce apoptosis prior to initiating infection, pathogenesis and evasion of host immune responses. In this study, we evaluated the efficacy of vaccinating chickens against Salmonella Enteritidis (SE) using a vaccine candidate strain (JOL919), constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. In present study day old chickens were inoculated with 1×10(7)cfu (colony forming unit) of JOL919 per os. We measured cell-mediated immunity, protective efficacy and extent of apoptosis induction in splenocytes. Seven days post-immunization, the number of CD3+CD4+ and CD3+ CD8+ T cells was significantly higher in the immunized group compared to the control group, indicating a significant augmentation of systemic immune response. The internal organs of chickens immunized with JOL919 had a significantly lower challenge-strain recovery, indicating effective protection and clearance of the challenge strain. Post-challenge, the number of apoptotic cells in the immunized group was significantly lower than in the control group. Additionally, AV/PI (Annexin V/propidium iodide) staining was performed to differentiate between apoptotic cells and necrotic cells, which corroborated TUNEL-assay (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) results. The proportions of AV+/PI- and AV+/PI+ cells, which represent the proportions of early apoptotic and late apoptotic/early necrotic cells present, respectively, were significantly lower in the immunized group. Our findings suggest that the apoptotic splenocytes in immunized chickens significantly decreased in number, which occurred concomitantly with a significant rise in systemic immune response and bacterial clearance. This suggests that inhibition of apoptosis may be a marker of protection efficacy in immunized chickens.

A Live Salmonella Gallinarum Vaccine Candidate Secreting an Adjuvant Protein Confers Enhanced Safety and Protection Against Fowl Typhoid.

Live attenuated vaccines are used for effective protection against fowl typhoid (FT) in domestic poultry. In this study, a lon/cpxR/asd deletion mutant of Salmonella Gallinarum expressing the B subunit of a heat labile toxin (LTB) from Escherichia coli, a known adjuvant, was cloned in a recombinant p15A ori plasmid, JOL1355, and evaluated as a vaccine candidate in chickens. The plasmid was shown to be stable inside the attenuated Salmonella Gallinarum cell after three successive generations. Moreover, from an environmental safety point of view, apart from day 1 the JOL1355 strain was not detected in feces through day 21 postinoculation. For the efficacy of JOL1355, a total of 100 chickens were equally divided into two groups. Group A (control) chickens were intramuscularly inoculated with phosphate-buffered saline at 4 and 8 wk of age. Group B chickens were primed and boosted via the intramuscular route with 200 µL of a bacterial suspension of JOL1355 containing 1 × 10(8) colony forming units. All the chickens in Group A and B were challenged at 3 wk postbooster by oral inoculation with a wild-type Salmonella Gallinarum strain, JOL420. The JOL1355-immunized group showed significant protection and survival against the virulent challenge compared to the nonimmunized group. In addition, Group B exhibited a significantly higher humoral immune response, and the chickens remained healthy without any symptoms of anorexia, diarrhea, or depression. Group B also exhibited a significantly lower mortality rate of 4% compared to the 46% of the control group, which can be attributed to higher immunogenicity and better protection. The Group B chickens had significantly lower lesion scores for affected organs, such as the liver and spleen, compared to those of the control chickens (P < 0.01). These findings suggest that JOL1355 is a promising candidate for a safe and highly immunogenic vaccine against FT.

Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry.

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Optical properties and laser damage threshold of HfO(2)-SiO(2) mixed composite thin films.

HfO(2)-SiO(2) mixed composite thin films have been deposited on fused silica substrate by co-evaporation of HfO(2) and SiO(2) through the reactive electron-beam evaporation technique. The composition-dependent refractive index and the absorption coefficient have been analyzed using different effective medium approximation (EMA) models in order to evaluate the suitability of these models for such mixed composite thin films. The discrepancies between experimentally determined and EMA-computed values are explained through microstructural and morphological evolutions observed in these mixed composite films. Finally, the dependence of the laser damage threshold as a function of silica content has been investigated, and the improved laser-induced damage threshold for films having more than 80% silica content has been explained through the defect-assisted multiphoton ionization process.

Growth factor expression pattern of homologous feeder layer for culturing buffalo embryonic stem cell-like cells.

The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages. Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-ß1, BMP-4 and GREMLIN but not of fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-ß1 (TGF-ß1), bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cell medium (SCM), SCM+1000IU mL(-1) leukemia inhibitory factor (LIF), SCM+5 ngmL(-1) FGF-2 or SCM+LIF+FGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-ß1 was similar in SCM and LIF+FGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cells on a feeder layer for 24 h, the TGF-ß1 concentration was higher with LIF+FGF-2 than with LIF or FGF-2 alone which, in turn, was higher than that in SCM. In the LIF+FGF-2 group, the concentration of TGF-ß1 was lower and that of ACTIVIN-A was higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signalling molecules that may help in self-renewal of buffalo ES cell-like cells.