Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

BACKGROUND: Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. RESULTS: First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. CONCLUSIONS: This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

Immunoproteomic identification of immunodominant antigens independent of the time of infection in Brucella abortus 2308-challenged cattle.

Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and predicted function. We suggest that the detection of prominent immunogenic proteins during the infection period can support the development of advanced diagnostic methods with high specificity and accuracy; subsidiarily, these proteins can provide supporting data to aid in developing novel vaccine candidates.

Interplay between clathrin and Rab5 controls the early phagocytic trafficking and intracellular survival of Brucella abortus within HeLa cells.

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.

Chemical stability of active ingredients in diluted veterinary disinfectant solutions under simulated storage conditions.

Introduction: The product labels of veterinary disinfectants specify their expiration dates to prevent the use of outdated products, as these may result in disinfection and biosecurity failures during outbreak situations. However, a clear standard for the storage conditions of diluted disinfectant solutions has not yet been established, and the effects of storage conditions have scarcely been investigated. To fill this research gap, our study examined the stability of the active ingredients of diluted veterinary disinfectants based on their change in concentrations when stored at various temperatures for various time periods. Methods: Twenty veterinary disinfectants effective against either foot-and-mouth disease or avian influenza viruses were selected. The disinfectants were diluted to effective concentrations following the manufacturer's instructions. Using selective analytical techniques, the concentrations of the active ingredients of the samples that had been stored for varying intervals at different temperatures (4, 20, 30, and 45°C) were determined. These samples included soaps and detergents, acids, oxidizing agents, aldehydes, and copper compounds. The active ingredient concentrations of two of the samples were determined following freezing/thawing cycle, to establish their stability when exposed to simulated winter conditions. Results: Our results showed that most of the active ingredients had concentrations of 90% or greater of their initial concentrations, indicating ≥90% stability over a 21-day period under the experimental storage conditions. However, there were some exceptions. Glutaraldehyde, formaldehyde, and malic acid are over 90% stable at ≤ 30°C for 21 days, but their concentrations decreased to below 90% of their initial concentrations at 45°C, indicating a decline in stability when stored at 45°C for 21 days. The concentrations of potassium peroxymonosulfate and peracetic acid rapidly declined with increasing time and temperature to less than 90% of their initial concentrations. Discussion: Based on our findings, we propose that diluted disinfectant solutions should preferably be prepared daily. However, if the daily preparation of a diluted disinfectant solution is not feasible, then our results can be used as a reference, providing basic scientific data on the chemical stability of diluted disinfectant solutions commonly used in the veterinary field, thus indicating suitable storage conditions.

Complete genome sequence of Brucella canis strain HSK A52141, isolated from the blood of an infected dog.

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.

Complete genome sequence of Brucella abortus A13334, a new strain isolated from the fetal gastric fluid of dairy cattle.

Brucella abortus is a major pathogen that infects livestock and humans. A new strain of B. abortus (A13334) was isolated from the fetal gastric fluid of a dairy cow, with the aim of using it to compare genetic properties, analyze virulence factor, and survey the epidemiological relationship to other Brucella species. Here, we report the complete and annotated genome sequence of B. abortus A13334.

Modified Vaccinia Virus Ankara as a Potential Biosafety Level 2 Surrogate for African Swine Fever Virus in Disinfectant Efficacy Tests.

In South Korea, despite the increase in emerging viral pathogens in the veterinary industry, only efficacy-tested, virus-specific disinfectants are allowed to be used. Moreover, domestic testing of disinfectants for their virucidal efficacies against foreign, malignant, infectious pathogens that are unreported within the country and/or contagious livestock diseases that require special attention regarding public hygiene are legally restricted. Therefore, the Animal and Plant Quarantine Agency (APQA) designed a study to select a potential biosafety level 2 surrogate of African swine fever virus (ASFV) for efficacy testing to improve the disinfectant approval procedures. For this, the modified vaccinia virus Ankara (MVA) was compared to ASFV in terms of its susceptibility to disinfectants. Effective concentrations of active substances of disinfectants (potassium peroxymonosulfate, sodium dichloroisocyanurate, malic acid, citric acid, glutaraldehyde, and benzalkonium chloride) against ASFV and MVA were compared; similarly, efficacies of APQA-listed commercial disinfectants were examined. Tests were performed according to APQA guidelines, and infectivities of ASFV and MVA were confirmed by hemadsorption and cytopathic effect, respectively. The results reveal that the disinfectants are effective against MVA at similar or higher concentrations than those against ASFV, validating the use of MVA as a potential biosafety level 2 surrogate for ASFV in efficacy testing of veterinary disinfectants.

Surrogate Selection for Foot-and-Mouth Disease Virus in Disinfectant Efficacy Tests by Simultaneous Comparison of Bacteriophage MS2 and Bovine Enterovirus Type 1.

In South Korea, testing disinfectants against foot-and-mouth disease virus (FMDV) that are contagious in livestock or that require special attention with respect to public hygiene can be manipulated only in high-level containment laboratories, which are not easily available. This causes difficulties in the approval procedure for disinfectants, such as a prolonged testing period. Additionally, the required biosafety level (BSL) in the case of FMDV has hindered its extensive studies. However, this drawback can be circumvented by using a surrogate virus to improve the performance of the efficacy testing procedure for disinfectants. Therefore, we studied bacteriophage MS2 (MS2) and bovine enterovirus type 1 (ECBO) with respect to disinfectant susceptibility for selecting a surrogate for FMDV according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. Effective concentrations of the active substances in disinfectants (potassium peroxymonosulfate, sodium dichloroisocyanurate, malic acid, citric acid, glutaraldehyde, and benzalkonium chloride) against FMDV, MS2, and ECBO were compared and, efficacies of eight APQA-listed commercial disinfectants used against FMDV were examined. The infectivity of FMDV and ECBO were confirmed by examination of cytopathic effects, and MS2 by plaque assay. The results reveal that the disinfectants are effective against MS2 and ECBO at higher concentrations than in FMDV, confirming their applicability as potential surrogates for FMDV in efficacy testing of veterinary disinfectants.

Advanced multiplex PCR assay for differentiation of Brucella species.

Two new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.

Virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus and avian influenza virus.

This study evaluated the virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus (ASFV) and avian influenza virus (AIV), according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. AEW (pH 5.0-6.5) was prepared using a commercially available "Electrolyzed Water Generator" with a free chlorine concentration (FCC) of 5-140 ppm, and its efficiency in reducing the titer of ASFV and AIV was tested in a suspension under low- and high-level organic soiling. Under low-level organic soiling conditions, AEW with FCC ≥40 ppm was effective against ASFV; under high-level organic soiling conditions, AEW with FCC ≥80 ppm was effective against ASFV. Under low-level organic soiling conditions, AEW with FCC ≥60 ppm was effective against AIV; under high-level organic soiling conditions, AEW with FCC ≥100 ppm was effective against AIV. The virucidal effect of AEW seemed dependent on the FCC and the presence of organic soiling. Based on these data, we recommend the following minimum FCCs in AEW treatment for routine disinfection in veterinary field under low- and high-level organic soiling conditions: for ASFV, 50 ppm and 100 ppm; and for AIV, 75 ppm and 125 ppm, respectively. In conclusion, the virucidal effects of AEW against ASFV and AIV emphasize its potential utility as a disinfectant, and we suggest considering organic soiling conditions while using AEW for implementing effective control measures for field applications.

Cross-Contamination of Enrofloxacin in Veterinary Medicinal and Nutritional Products in Korea.

Poultry meat and eggs are vital sources of protein for human consumption worldwide. The use of several nutritional and medicinal products, including antibiotics, is crucial for efficient and safe poultry production. Accumulation of drug residues in meat and eggs from inappropriate drug use is a major concern to public health. Recently, enrofloxacin was detected (2.4-3.8 ppb) in edible eggs produced in Jeju Island, Korea. Although the farm from which the enrofloxacin-contaminated eggs were collected did not use enrofloxacin-containing products, they reported extensive use of a nutritional product (NPJ). Accordingly, in this study, we investigated whether enrofloxacin contamination had occurred accidentally in various widely used veterinary pharmaceutical products. Enrofloxacin content (4.57-179.08 ppm) in different lots of the NPJ was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Furthermore, 76 veterinary pharmaceutical products that are widely used in poultry farms in Korea and claim to not contain enrofloxacin were collected and analyzed by LC-MS/MS. Among them, a florfenicol product and a sulfatrimethoprime product were found to contain 3.00 and 0.57 ppm enrofloxacin, respectively. These results suggest that appropriate manufacturing standards are not being followed and that strict monitoring of drug manufacturing is necessary in Korea to avoid drug contamination.

Characterization of antimicrobial resistance of recent Salmonella enterica serovar Gallinarum isolates from chickens in South Korea.

Salmonella enterica serovar Gallinarum isolates (n=105) from chickens in South Korea between 2002 and 2007 were tested for antimicrobial susceptibility by determining minimum inhibitory concentrations of 16 antimicrobials, and their predominant resistance profiles were genetically characterized. Most isolates (99/105; 94.3%) were resistant to nalidixic acid and resistant/intermediately resistant to fluoroquinolones, and 63.8% (67/105) of the isolates were resistant to three or more antimicrobials. Forty-two quinolone-resistant isolates, of which the quinolone resistance-determining regions of the gyrA genes were sequenced, contained a substitution of a Ser to a Phe or Tyr at position 83 (71.4%), or a substitution of an Asp to an Asn, Gly, or Tyr at position 87 (28.6%). Fifty-seven sulphamethoxazole-resistant isolates were tested for the presence of class 1 integrons by polymerase chain reaction, and their resistance gene cassettes were analysed by sequencing. Three different class 1 integrons containing the resistance-gene insert aadA (52.6%; n=30), aadB (12.3%; n=7), or aadB-aadA (12.3%; n=7) were identified. Most isolates harbouring the integron containing aadB-aadA displayed resistance to all three aminoglycosides tested and also showed increased resistance to fluoroquinolones. These findings suggest that fluoroquinolone resistance may be epidemiologically linked to multiple aminoglycoside resistance.

Synergism of the Combination of Traditional Antibiotics and Novel Phenolic Compounds against Escherichia coli.

Pathogenic Escherichia coli (E. coli)-associated infections are becoming difficult to treat because of the rapid emergence of antibiotic-resistant strains. Novel approaches are required to prevent the progression of resistance and to extend the lifespan of existing antibiotics. This study was designed to improve the effectiveness of traditional antibiotics against E. coli using a combination of the gallic acid (GA), hamamelitannin, epicatechin gallate, epigallocatechin, and epicatechin. The fractional inhibitory concentration index (FICI) of each of the phenolic compound-antibiotic combinations against E. coli was ascertained. Considering the clinical significance and FICI, two combinations (hamamelitannin-erythromycin and GA-ampicillin) were evaluated for their impact on certain virulence factors of E. coli. Finally, the effects of hamamelitannin and GA on Rattus norvegicus (IEC-6) cell viability were investigated. The FICIs of the antibacterial combinations against E. coli were 0.281-1.008. The GA-ampicillin and hamamelitannin-erythromycin combinations more effectively prohibited the growth, biofilm viability, and swim and swarm motilities of E. coli than individual antibiotics. The concentration of hamamelitannin and GA required to reduce viability by 50% (IC50) in IEC-6 cells was 988.54 µM and 564.55 µM, correspondingly. GA-ampicillin and hamamelitannin-erythromycin may be potent combinations and promising candidates for eradicating pathogenic E. coli in humans and animals.

Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea.

BACKGROUND: A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates. RESULTS: A total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the Brucella isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via in-vitro and in-vivo passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign Brucella isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates. CONCLUSION: The MLVA assay has enough discrimination power in the Brucella species level and can be utilized as a tool for the epidemiological trace-back of the B. abortus isolates. But it is important to consider that Brucella isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.

Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea.

During the 2006-2007 winter season in South Korea, several outbreaks of highly pathogenic avian influenza virus (H5N1) were confirmed among domestic poultry and in migratory bird habitats. Phylogenetic analysis showed that all isolates were closely related and that all belong to the A/bar-headed goose/Qinghai/5/2005-like lineage rather than the A/chicken/Korea/ES/2003-like lineage.

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries.

First Isolation of Streptococcus halichoeri and Streptococcus phocae from a Steller Sea Lion (Eumetopias jubatus) in South Korea.

Streptococcus species are emerging potential pathogens in marine mammals. We report the isolation and identification of Streptococcus halichoeri and Streptococcus phocae in a Steller sea lion (Eumetopias jubatus) in South Korea.

Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms.

To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.

Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/µl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field.