scholarly journals Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260288
Author(s):  
Carlos Chacón-Díaz ◽  
Ana Zabalza-Baranguá ◽  
Beatriz San Román ◽  
José-María Blasco ◽  
Maite Iriarte ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Fluorescent Protein ◽  
Indirect Elisa ◽  
Economic Losses ◽  
Bovine Brucellosis ◽  
Serological Identification ◽  
Green Fluorescent ◽  
Chromosome Ii ◽  
The Rose ◽  
S19 Vaccine

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals eroprevalence of bovine brucellosis in trade cattle slaughtered in Ibadan, Nigeria, from 2004-2006

2010 ◽  
Vol 81 (1) ◽  
Author(s):  
S. I.B. Cadmus ◽  
H. K. Adesokan ◽  
B. O. Adedokun ◽  
J. A. Stack
Keyword(s):  
Brucella Abortus ◽  
Age Distribution ◽  
Bovine Brucellosis ◽  
Southwestern Nigeria ◽  
The Public ◽  
Seroprevalence Study ◽  
Significant Difference ◽  
The Rose ◽  
Ibadan Nigeria

A seroprevalence study was carried out among trade cattle slaughtered at Bodija Municipal Abattoir, Ibadan (southwestern Nigeria) over a period of 3 consecutive years from 2004 to 2006 with a view to determining the breed, sex and age distribution in the seropositivity of bovine brucellosis. In total, 1642 animals were examined for antibodies to Brucella abortus using the Rose Bengal test. Seroprevalences of 6.00 %, 6.17 % and 5.31 % were obtained in the years 2004, 2005 and 2006, respectively but a decrease in 2006 shows no significant difference (P>0.05). The role of the breed (P>0.05), sex (P>0.05) and age (P>0.05) in the occurrence of the infection was not statistically significant at 5 %, although higher rates were obtained for females and older animals. The trend in the disease over the 3-year period showed that it is endemic in trade cattle slaughtered in Ibadan and the public health implications of this are discussed.

scholarly journals Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

2021 ◽  
Vol 12 ◽  
Author(s):  
Qing Pan ◽  
Yu Zhang ◽  
Aijing Liu ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Virus Genome ◽  
Open Reading Frames ◽  
Vaccine Vector ◽  
Economic Losses ◽  
Foreign Gene ◽  
The Novel ◽  
Hydropericardium Syndrome ◽  
Fowl Adenovirus ◽  
Green Fluorescent

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

Detection and localization of the endophyte Undifilum oxytropis in locoweed tissues

Botany ◽  
2012 ◽  
Vol 90 (12) ◽  
pp. 1229-1236 ◽  
Author(s):  
Roxanna Reyna ◽  
Peter Cooke ◽  
Daniel Grum ◽  
Daniel Cook ◽  
Rebecca Creamer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Growth Patterns ◽  
Host Cells ◽  
Economic Losses ◽  
Thin Sections ◽  
Oxytropis Sericea ◽  
Pathogenic Interaction ◽  
Green Fluorescent ◽  
Detection And Localization

Poisoning of livestock owing to grazing on locoweeds results in significant economic losses in the western United States. Some Oxytropis spp. locoweeds contain a seed-transmitted endophytic fungus, Undifilum oxytropis, which produces the toxic alkaloid swainsonine. We sought to localize and characterize growth patterns of the fungus within leaves and petioles of Oxytropis lambertii Pursh and Oxytropis sericea Nutt. to help define the types of interactions between the fungus and its hosts. Vegetative hyphae were observed within locoweed tissues using integrated imaging. Topographical images from scanning electron microscopy revealed the presence of the endophyte in the pith tissue of petioles. The fungus was identified between plant cells but did not appear to penetrate host cells. Transmission electron microscopy images of thin sections revealed that hyphae were closely associated with host cell walls. Oxytropis sericea was innoculated with green fluorescent protein-transformed U. oxytropis and observed by confocal microscopy, confirming the presence of the endophyte hyphae in leaves and petioles. The fungus was identified only in the pith of petioles using fluorescence and in the vascular bundle throughout extracellular spaces in leaves. These results revealed no signs of a pathogenic interaction between plant and fungus and support the hypothesis of a mutualistic or commensal relationship.

scholarly journals Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

2015 ◽  
Vol 83 (4) ◽  
pp. 1458-1464 ◽  
Author(s):  
Job Alves de Souza Filho ◽  
Vicente de Paulo Martins ◽  
Priscila Carneiro Campos ◽  
Juliana Alves-Silva ◽  
Nathalia V. Santos ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Economic Losses ◽  
Macrophage Infection ◽  
Content Type ◽  
Live Vaccines ◽  
Interferon Regulatory Factor 1 ◽  
Potential Vaccine ◽  
Green Fluorescent

Brucellaspecies can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence ofBrucellaspp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of theBrucellamembrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed thatB. abortusΔmfp::kanand Δomp19::kandeletion mutant strains have reduced persistencein vivoin C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kanor Δomp19::kanstrain expressing green fluorescent protein (GFP) approximately 80% or 65% ofBrucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments.B. abortusΔomp19::kanwas attenuatedin vivobut had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kanstrain had a lower virulence in these same mouse models. Furthermore, Δmfp::kanand Δomp19::kanstrains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kanstrain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kanstrain induced protection similar to that of RB51. Thus, these results demonstrate thatBrucellaMfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kanmutant may serve as a potential vaccine candidate in future studies.

scholarly journals Brucella abortus Genes Identified following Constitutive Growth and Macrophage Infection

2001 ◽  
Vol 69 (12) ◽  
pp. 7736-7742 ◽  
Author(s):  
Linda Eskra ◽  
Aurea Canavessi ◽  
Merriann Carey ◽  
Gary Splitter
Keyword(s):  
Brucella Abortus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Initiation Factor ◽  
Gene Repair ◽  
Reporter System ◽  
Macrophage Infection ◽  
Regulatory Systems ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems. Our goal is to develop a vaccine for Brucella. To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B. abortusgenes. Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance). Of greater interest, clones negative for constitutively producedgfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection. Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions. Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K+ transport gene), and site-specific recombination (xerD gene). A gene involved in metabolism and biosynthesis (deoxyxylulose 5′ phosphate synthase gene) was also identified. Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism). Identification of B. abortusgenes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.

scholarly journals Evaluation of an Indirect ELISA for the Diagnosis of Bovine Brucellosis

1995 ◽  
Vol 7 (4) ◽  
pp. 473-475 ◽  
Author(s):  
Francisco A. Uzal ◽  
Alejandra E. Carrasco ◽  
Susana Echaide ◽  
Klaus Nielsen ◽  
Carlos A. Robles
Keyword(s):  
Indirect Elisa ◽  
Screening Tests ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Brucellosis ◽  
Energy Agency ◽  
Elisa Kit ◽  
Large Numbers ◽  
The Rose ◽  
Epidemiological Information ◽  
Control And Eradication

Control and eradication of bovine brucellosis is usually based on the serological detection of antibodies. In Argentina, the rose bengal test (RB) and the buffered plate antigen test (BPA) are the 2 screening tests officially recognized, whereas the 2-mercaptoethanol test (2ME) and the tube agglutination test (SAT) are the confirmatory assays currently in use. In order to improve the serological diagnosis of bovine brucellosis in Patagonia, Argentina, an indirect enzyme-linked immunosorbent assay (I-ELISA) kit distributed by the International Atomic Energy Agency was evaluated. Sera from negative nonvaccinated, negative but vaccinated, and positive animals were tested by all the above techniques. The specificity of the I-ELISA (98.9% and 97.2%) was similar to that of the BPA, RB, 2ME and complement fixation (CF) tests when used to test sera from negative nonvaccinated and negative but vaccinated animals, respectively. The sensitivity of the I-ELISA (98.7%) was higher than the BPA test (96.1%) and the CF test (95.2%). The I-ELISA kit evaluated in this study was thought to be a valuable tool for the diagnosis of bovine brucellosis in the Patagonia region where little epidemiological information is available about this disease, and where large numbers of sera should be tested to obtain such information.

scholarly journals Estimating the probability of freedom from bovine brucellosis in the Galapagos Islands

2018 ◽  
Vol 147 ◽  
Author(s):  
G. Gioia ◽  
R. L. Vinueza ◽  
M. Cruz ◽  
M. Jay ◽  
Y. Corde ◽  
...  
Keyword(s):  
Latin America ◽  
Indirect Elisa ◽  
Galapagos Islands ◽  
Galápagos Islands ◽  
Bovine Brucellosis ◽  
Santa Cruz ◽  
Negative Results ◽  
Level Design ◽  
Cattle Farms ◽  
The Rose

AbstractBovine brucellosis is a worldwide zoonotic disease that still burdens several countries in the Mediterranean, Asia, Africa and Latin America. Although the disease is present in Ecuador, the Galapagos Islands seem to be free from the disease based on a survey conducted in 1997 where all tested animals showed negative results. This study aimed at estimating the probability of freedom from brucellosis in this Ecuadorian province in 2014. A survey was implemented on the three main cattle-producing islands of the province: Santa Cruz, Isabela and San Cristóbal. Thirty-three cattle farms and 410 cattle were tested for brucellosis using the Rose Bengal test and indirect ELISA. All animals showed negative results for both tests. Probability of freedom was estimated at 98%, 91% and 88% for Santa Cruz, Isabela and San Cristóbal, respectively, considering a herd-level design seroprevalence of 20% and animal-level design seroprevalence of 15%, and assuming a perfect specificity of the survey. The negative results found in 1997 and present surveys suggest that the Galapagos Islands are free from bovine brucellosis.

scholarly journals Brucella abortus Strain RB51 Leucine Auxotroph as an Environmentally Safe Vaccine for Plasmid Maintenance and Antigen Overexpression

2008 ◽  
Vol 74 (22) ◽  
pp. 7051-7055 ◽  
Author(s):  
Parthiban Rajasekaran ◽  
Mohamed N. Seleem ◽  
Andrea Contreras ◽  
Endang Purwantini ◽  
Gerhardt G. Schurig ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Brucella Abortus ◽  
Fluorescent Protein ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Heterologous Antigen ◽  
Safe Vaccine ◽  
Environmentally Safe ◽  
Cd1 Mice ◽  
Green Fluorescent

ABSTRACT To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

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