Comparative Analysis of Antibiotic Resistance and Biofilm Characteristics of Two Major Enterococcus Species from Poultry Slaughterhouses in South Korea.

The spread of antibiotic-resistant Enterococcus in the poultry industry poses significant public health challenges due to multidrug resistance and biofilm formation. We investigated the antibiotic resistance profiles and biofilm characteristics of E. faecalis and E. faecium isolates from chicken meat in poultry slaughterhouses in South Korea. Ninety-six isolates (forty-eight each of E. faecalis and E. faecium) were collected between March and September 2022. Both species were analyzed using MALDI-TOF, PCR, antibiotic susceptibility testing, and biofilm assays. A high level of multidrug resistance was observed in E. faecalis (95.8%) and E. faecium (93.8%), with E. faecium exhibiting a broader range of resistance, particularly to linezolid (52.1%) and rifampicin (47.9%). All E. faecalis isolates formed biofilm in vitro, showing stronger biofilm formation than E. faecium with a significant difference (p < 0.001) in biofilm strength. Specific genes (cob, ccf, and sprE) were found to be correlated with biofilm strength. In E. faecium isolates, biofilm strength was correlated with resistance to linezolid and rifampicin, while a general correlation between antibiotic resistance and biofilm strength was not established. Through analysis, correlations were noted between antibiotics within the same class, while no general trends were evident in other analyzed factors. This study highlights the public health risks posed by multidrug-resistant enterococci collected from poultry slaughterhouses, emphasizing the complexity of the biofilm-resistance relationship and the need for enhanced control measures.

Genetic Characterization and Phylogeographic Analysis of the First H13N6 Avian Influenza Virus Isolated from Vega Gull in South Korea.

Avian influenza virus (AIV) is a pathogen with zoonotic and pandemic potential. Migratory birds are natural reservoirs of all known subtypes of AIVs, except for H17N10 and H18N11, and they have been implicated in previous highly pathogenic avian influenza outbreaks worldwide. This study identified and characterized the first isolate of the H13N6 subtype from a Vega gull (Larus vegae mongolicus) in South Korea. The amino acid sequence of hemagglutinin gene showed a low pathogenic AIV subtype and various amino acid substitutions were found in the sequence compared to the reference sequence and known H13 isolates. High sequence homology with other H13N6 isolates was found in HA, NA, PB1, and PA genes, but not for PB2, NP, M, and NS genes. Interestingly, various point amino acid mutations were found on all gene segments, and some are linked to an increased binding to human-type receptors, resistance to antivirals, and virulence. Evolutionary and phylogenetic analyses showed that all gene segments are gull-adapted, with a phylogeographic origin of mostly Eurasian, except for PB2, PA, and M. Findings from this study support the evidence that reassortment of AIVs continuously occurs in nature, and migratory birds are vital in the intercontinental spread of avian influenza viruses.

Development of a Parturition Detection System for Korean Native Black Goats.

Korean Native Black Goats deliver mainly during the cold season. However, in winter, there is a high risk of stunted growth and mortality for their newborns. Therefore, we conducted this study to develop a KNBG parturition detection system that detects and provides managers with early notification of the signs of parturition. The KNBG parturition detection system consists of triaxial accelerometers, gateways, a server, and parturition detection alarm terminals. Then, two different data, the labor and non-labor data, were acquired and a Decision Tree algorithm was used to classify them. After classifying the labor and non-labor states, the sum of the labor status data was multiplied by the activity count value to enhance the classification accuracy. Finally, the Labor Pain Index (LPI) was derived. Based on the LPI, the optimal processing time window was determined to be 10 min, and the threshold value for labor classification was determined to be 14 240.92. The parturition detection rate was 82.4%, with 14 out of 17 parturitions successfully detected, and the average parturition detection time was 90.6 min before the actual parturition time of the first kid. The KNBG parturition detection system is expected to reduce the risk of stunted growth and mortality due to hypothermia in KNBG kids by detecting parturition 90.6 min before the parturition of the first kid, with a success rate of 82.4%, enabling parturition nursing.

Intracellular Growth Inhibition and Host Immune Modulation of 3-Amino-1,2,4-triazole in Murine Brucellosis.

Catalase, an antioxidant enzyme widely produced in mammalian cells and bacteria, is crucial to mitigating oxidative stress in hostile environments. This function enhances the intracellular survivability of various intracellular growth pathogens, including Brucella (B.) abortus. In this study, to determine whether the suppression of catalase can inhibit the intracellular growth of B. abortus, we employed 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, in both RAW 264.7 macrophage cells and an ICR mouse model during Brucella infection. The intracellular growth assay indicated that 3-AT exerts growth-inhibitory effects on B. abortus within macrophages. Moreover, it contributes to the accumulation of reactive oxygen species and the formation of nitric oxide. Notably, 3-AT diminishes the activation of the nucleus transcription factor (NF-κB) and modulates the cytokine secretion within infected cells. In our mouse model, the administration of 3-AT reduced the B. abortus proliferation within the spleens and livers of infected mice. This reduction was accompanied by a diminished immune response to infection, as indicated by the lowered levels of TNF-α, IL-6, and IL-10 and altered CD4+/CD8+ T-cell ratio. These results suggest the protective and immunomodulatory effects of 3-AT treatment against Brucella infection.

Promotion of Th1 and Th2 responses over Th17 in Riemerella anatipestifer stimulation in chicken splenocytes: Correlation of gga-miR-456-3p and gga-miR-16-5p with NOS2 and CCL5 expression.

Riemerella (R.) anatipestifer poses a significant threat to ducks, resulting in mortality rates ranging from 5-75%. This disease is highly infectious and economically consequential for domestic ducks. Although other avian species, such as chickens, also display susceptibility, the impact is comparatively less severe than in ducks. IL-17A has a pronounced correlation with R. anatipestifer infection in ducks, which is less in chickens. This study performed an in vitro transcriptome analysis using chicken splenic lymphocytes collected at 4-, 8-, and 24-hour intervals following R. anatipestifer stimulation. The primary objective was to discern the differentially expressed genes, with a specific focus on IL-17A and IL-17F expression. Moreover, an association between specific miRNAs with NOS2 and CCL5 was identified. The manifestation of riemerellosis in chickens was linked to heightened expression of Th1- and Th2-associated cells, while Th17 cells exhibited minimal involvement. This study elucidated the mechanism behind the absence of a Th17 immune response, shedding light on its role throughout disease progression. Additionally, through small RNA sequencing, we identified a connection between miRNAs, specifically miR-456-3p and miR-16-5p, and their respective target genes NOS2 and CCL5. These miRNAs are potential regulators of the inflammatory process during riemerellosis in chickens.

The Modulatory Effect of Sodium Propionate Treatment in the Expression of Inflammatory Cytokines and Intracellular Growth of Brucella abortus 544 in Raw 264.7 Cells.

In this study, we investigated the effects of sodium propionate (SP) treatment on intracellular mechanism of murine macrophages and its contribution to host immunity during Brucella abortus 544 infection. The intracellular growth assay revealed that SP inhibited Brucella replication inside the macrophages. To determine intracellular signaling involved during SP treatment after Brucella infection, we analyzed the change of five different cytokines production relevant to SP such as TNF-α, IL-10, IFN-γ, IL-1ß, and IL-6, and the results indicated that the boost with IL-10 was apparent throughout the culture period for 48 h as well as IL-1ß which was apparent at 24 h post-infection and IFN-γ which was apparent at 24 h and 48 h in comparison to SP untreated groups. On the other way, SP-treated cells displayed suppressed production of TNF-α and IL-6 at all time points tested and 48 h post-infection, respectively. Furthermore, we conducted western blot to establish a cellular mechanism, and the result suggested that SP treatment attenuated p50 phosphorylation, part of the NF-κB pathway. These findings indicated that the inhibitory effect of SP against Brucella infection could be attributed through induction of cytokine production and interference on intracellular pathway, suggesting SP as a potential candidate for treating brucellosis.

Protective Effects against Brucella abortus 544 Infection in a Murine Macrophage Cell Line and in a Mouse Model via Treatment with Sirtuin 1 Activators Resveratrol, Piceatannol and Ginsenoside Rg3.

Brucellosis is a contagious zoonotic disease that infects millions of people annually with hundreds of millions more being exposed. It is caused by Brucella, a highly infectious bacterial species capable of infecting humans with an estimated dose of 10-100 organisms. Sirtuin 1 (SIRT1) has been reported to contribute to prevention of viral diseases as well as a chronic infection caused by Mycobacterium bovis. Here, we investigated the role of SIRT1 in the establishment of Brucella abortus infection in both in vitro and in vivo systems using the reported SIRT1 activators resveratrol (RES), piceatannol (PIC), and ginsenoside Rg3 (Rg3). In RAW264.7 cells, SIRT1 activators did not alter the adherence of Brucella or Salmonella Typhimurium. However, reduced uptake of Brucella was observed in cells treated with PIC and Rg3, and survival of Brucella within the cells was only observed to decrease in cells that were treated with Rg3, while PIC treatment reduced the intracellular survival of Salmonella. SIRT1 treatment in mice via oral route resulted in augmented Brucella resistance for PIC and Rg3, but not RES. PIC treatment favors Th2 immune response despite reduced serum proinflammatory cytokine production, while Rg3-treated mice displayed high IL-12 and IFN-γ serum production. Overall, our findings encourage further investigation into the complete mechanisms of action of the different SIRT1 activators used as well as their potential benefit as an effective alternative approach against intracellular and extracellular pathogens.

Comparative analysis of evaluation parameters in E. acervulina, E. maxima and E. tenella-infected broilers.

Three parameters, body weight gain (BWG), intestinal lesion score (LS) and fecal oocyst shedding, were compared in broilers infected with major parasitic species; Eimeria acervulina, E. maxima, and E. tenella. First, two- and three-week-old chickens with Eimeria infection showed LS of approximately 3, but two-week-old chickens were more correlated with BWG. Second, significant differences in BWG were observed between male and female broilers challenged with Eimeria. Finally, E. maxima-infected broilers among three Eimeria species showed a higher relationship between BWG and LS, suggesting three considerations such as genders, age and Eimeria species for Eimeria experiments.

The In Vitro and In Vivo Effect of Lipoxygenase Pathway Inhibitors Nordihydroguaiaretic Acid and Its Derivative Tetra-O-methyl Nordihydroguaiaretic Acid against Brucella abortus 544.

This study investigated the contribution of lipoxygenase (LOX) inhibitors, nordihydroguaiaretic acid (NDGA), tetra-O-methyl nordihydroguaiaretic acid (M4N) and zileuton (ZIL), and thromboxane A2 (TXA2) inhibitor 4,5-diphenylimidazole (DPI) in the proliferation of Brucella abortus infection. None of the compounds affected the uptake of Brucella into the macrophages. We determined the effect of neutralizing leukotriene B4 (LTB4) receptor and showed that the uptake of the bacteria was inhibited at 30 min post-infection. M4N treatment attenuated intracellular survival of Brucella at 2 h post-incubation but it was not observed in the succeeding time points. DPI treatment showed reduced survival of Brucella at 24 h post-incubation while blocking LTB4 receptor was observed to have a lower intracellular growth at 48 h post-incubation suggesting different action of the inhibitors in the course of the survival of Brucella within the cells. Reduced proliferation of the bacteria in the spleens of mice was observed in animals treated with ZIL or DPI. Increased serum cytokine level of TNF-α and MCP-1 was observed in mice treated with M4N or ZIL while a lower IFN-γ level in ZIL-treated mice and a higher IL-12 serum level in DPI-treated mice were observed at 7 d post-infection. At 14 d post-infection, ZIL-treated mice displayed reduced serum level of IL-12 and IL-10. Overall, inhibition of 5-LOX or TXA2 or a combination therapy promises a potential alternative therapy against B. abortus infection. Furthermore, strong ligands for LTB4 receptor could also be a good candidate for the control of Brucella infection.

Simvastatin Inhibits Brucella abortus Invasion into RAW 264.7 Cells through Suppression of the Mevalonate Pathway and Promotes Host Immunity during Infection in a Mouse Model.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and has been found to have protective effects against several bacterial infections. In this study, we investigate the effects of simvastatin treatment on RAW 264.7 macrophage cells and ICR mice against Brucella (B.) abortus infections. The invasion assay revealed that simvastatin inhibited the Brucella invasion into macrophage cells by blocking the mevalonic pathway. The treatment of simvastatin enhanced the trafficking of Toll-like receptor 4 in membrane lipid raft microdomains, accompanied by the increased phosphorylation of its downstream signaling pathways, including JAK2 and MAPKs, upon =Brucella infection. Notably, the suppressive effect of simvastatin treatment on Brucella invasion was not dependent on the reduction of cholesterol synthesis but probably on the decline of farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthesis. In addition to a direct brucellacidal ability, simvastatin administration showed increased cytokine TNF-α and differentiation of CD8+ T cells, accompanied by reduced bacterial survival in spleens of ICR mice. These data suggested the involvement of the mevalonate pathway in the phagocytosis of B. abortus into RAW 264.7 macrophage cells and the regulation of simvastatin on the host immune system against Brucella infections. Therefore, simvastatin is a potential candidate for studying alternative therapy against animal brucellosis.

Epidemiological investigation and drug resistance of Eimeria species in Korean chicken farms.

BACKGROUND: Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. RESULTS: An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. CONCLUSION: The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.

Cobalt (II) Chloride Regulates the Invasion and Survival of Brucella abortus 544 in RAW 264.7 Cells and B6 Mice.

The effects of Cobalt (II) chloride (CoCl2) in the context of Brucella abortus (B. abortus) infection have not been evaluated so far. Firstly, we found that CoCl2 treatment inhibited the phagocytosis of B. abortus into RAW 264.7 cells. The inhibition of bacterial invasion was regulated by F-actin formation and associated with a reduction in the phosphorylation of ERK1/2 and HIF-1α expression. Secondly, the activation of trafficking regulators LAMP1, LAMP2, and lysosomal enzyme GLA at the transcriptional level activated immune responses, weakening the B. abortus growth at 4 h post-infection (pi). The silencing of HIF-1α increased bacterial survival at 24 h pi. At the same time, CoCl2 treatment showed a significant increase in the transcripts of lysosomal enzyme HEXB and cytokine TNF-α and an attenuation of the bacterial survival. Moreover, the enhancement at the protein level of HIF-1α was induced in the CoCl2 treatment at both 4 and 24 h pi. Finally, our results demonstrated that CoCl2 administration induced the production of serum cytokines IFN-γ and IL-6, which is accompanied by dampened Brucella proliferation in the spleen and liver of treated mice, and reduced the splenomegaly and hepatomegaly. Altogether, CoCl2 treatment contributed to host resistance against B. abortus infection with immunomodulatory effects.

Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection.

Riemerella anatipestifer is one of the most devastating pathogens affecting the global duck farms. Infection is involved in secretion of proinflammatory cytokines, including interleukin- (IL-) 17A. During the immune response to infection, IL-22 and IL-17A are often produced concurrently and at high levels in inflamed tissues. Little is known about duck IL-22 (duIL-22) during R. anatipestifer infection. We describe the characterization of duIL-22 and its mRNA expression analysis in splenic lymphocytes and macrophages treated with heat-killed R. anatipestifer and in the spleens and livers of R. anatipestifer-infected ducks. Full-length cDNA of duIL-22 encoded 197 amino acids. The deduced amino acid sequence of duIL-22 shared a 30.4-40.5% similarity with piscine counterparts, 57.4-60.1% with mammalian homologs, and 93.4% similarity to the chicken. Duck IL-22 mRNA expression level was relatively high in the skin of normal ducks. It was increased in mitogen-stimulated splenic lymphocytes and in killed R. anatipestifer-activated splenic lymphocytes and macrophages. Compared with healthy ducks, IL-22 transcript expression was significantly upregulated in the livers and spleens on days 1 and 4 postinfection, but not on day 7. IL-17A was significantly increased in the spleens only on day 4 postinfection and in the livers at all time points. When splenic lymphocytes were stimulated with heat-killed R. anatipestifer, CD4+ cells predominantly produced IL-22 while IL-17A was expressed both by CD4+ and CD4- cells. These results suggested that IL-22 and IL-17A are likely expressed in different cell types during R. anatipestifer infection.

Expression of Chicken NK-Lysin and Its Role in Chicken Coccidiosis Induced by Eimeria necatrix.

Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis.

Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella.

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Anticoccidial Activity of Berberine against Eimeria-Infected Chickens.

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.

Immune-metabolic receptor GPR84 surrogate and endogenous agonists, 6-OAU and lauric acid, alter Brucella abortus 544 infection in both in vitro and in vivo systems.

Brucella abortus, one of the most important members of the genus Brucella responsible for human disease, is an intracellular pathogen capable of avoiding or interfering components of the host immune responses that are critical for its virulence. GPR84, on the other hand, is a seven-transmembrane GPCR involved in the inflammatory response and its induced expression was associated with B. abortus infection of RAW264.7 cells. Here we examined the effects of the reported GPR84 surrogate and endogenous agonists, namely 6-n-octylaminouracil (6-OAU) and lauric acid (LU), respectively in the progression of B. abortus infection in a cell and mouse models. The in vitro studies revealed the LU had bactericidal effect against Brucella starting at 24 h post-incubation. Adhesion of Brucella to RAW264.7 cells was attenuated in both 6-OAU and LU treatments. Brucella uptake was observed to be inhibited in a dose and time-dependent manner in 6-OAU but only at the highest non-cytotoxic concentration in LU-treated cells. However, survival of Brucella within the cells was reduced only in LU-treated cells. We also investigated the possible inhibitory effects of the agonist in other Gram-negative bacterium, Salmonella Typhimurium and we found that both adhesion and uptake were inhibited in 6-OAU treatment and only the intracellular survival for LU treatment. Furthermore, 6-OAU treatment reduced ERK phosphorylation and MCP-1 secretion during Brucella infection as well as reduced MALT1 protein expression and ROS production in cells without infection. LU treatment attenuated ERK and JNK phosphorylation, MCP-1 secretion and NO accumulation but increased ROS production during infection, and similar pattern with MALT1 protein expression. The in vivo studies showed that both treatments via oral route augmented resistance to Brucella infection but more pronounced with 6-AOU as observed with reduced bacterial proliferation in spleens and livers. At 7 d post-treatment and 14 d post-infection, 6-OAU-treated mice displayed reduced IFN-γ serum level. At 7 d post-infection, high serum level of MCP-1 was observed in both treatments with the addition of TNF-α in LU group. IL-6 was increased in both treatments at 14 d post-infection with higher TNF-α, MCP-1 and IL-10 in LU group. Taken together, 6-OAU and LU are potential candidates representing pharmaceutical strategy against brucellosis and possibly other intracellular pathogens or inflammatory diseases.

Formyl peptide receptor 2 (FPR2) antagonism is a potential target for the prevention of Brucella abortus 544 infection.

Here, we explore the potential role of formyl peptide receptor 2 (FPR2) during Brucella abortus infection. FPR2 manipulation affected B. abortus internalization but not its growth within macrophages. During the activation of FPR2 induced by its agonist AGP-8694, a high level of Brucella uptake was accompanied by an increase in ERK phosphorylation, while intracellular survival at 24 h postincubation was observed to be associated with slightly reduced nitrite accumulation but augmented superoxide anion production. Attenuated secretion of IL-6 and IL-10 were observed 48 h postincubation in the bone marrow-derived macrophages (BMDMs) treated with the FPR2 antagonist WRW4. An opposite pattern of bacterial uptake was observed upon treatment with the FPR2 antagonist, but no significant changes in the activation of MAPKs or the production of nitrite or superoxide anion were observed. Interestingly, AGP-8694 treatment of mice did not lead to differences in spleen or liver weight but slightly enhanced bacterial proliferation was observed in the spleen. Although the weights of the spleen or liver did not differ, WRW4 treatment led to reduced bacterial proliferation in the spleen. Furthermore, FPR2 antagonist treatment was associated with high serum levels of the proinflammatory cytokines IL-12, TNF-α, IFN-γ and MCP-1, while the production of TNF-α was inhibited in AGP-8694-treated mice. IL-6 and IL-10 levels were slightly increased in AGP-8694-treated mice at 24 h postinfection. Our findings demonstrated the contribution of FPR2 via manipulating this receptor using its reported agonist AGP-8694 and antagonist WRW4 in both in vitro and in vivo systems. Although activation of the receptor did not consistently induced Brucella infection, FPR2 inhibition may be a promising strategy to treat brucellosis in animals which encourages further investigation.

Prostaglandin I2 (PGI2) inhibits Brucella abortus internalization in macrophages via PGI2 receptor signaling, and its analogue affects immune response and disease outcome in mice.

To date, the implications of prostaglandin I2 (PGI2), a prominent lipid mediator for modulation of immune responses, has not been clearly understood in Brucella infection. In this study, we found that cyclooxygenase-2 (COX-2) was significantly expressed in both infected bone marrow-derived macrophages (BMMs) and RAW 264.7 cells. Prostaglandin I2 synthase (PTGIS) expression was not significantly changed, and PGI2receptor (PTGIR) expression was downregulated in BMMs but upregulated in RAW 264.7 macrophages at late infection. Here, we presented that PGI2, a COX-derived metabolite, was produced by macrophages during Brucella infection and its production was regulated by COX-2 and IL-10. We suggested that PGI2 and selexipag, a potent PGI2 analogue, inhibited Brucella internalization through IP signaling which led to down-regulation of F-actin polymerization and p38α MAPK activity. Administration with selexipag suppressed immune responses and resulted in a notable reduction in bacterial burden in spleen of Brucella-challenged mice. Taken together, our study is the first to characterize PGI2 synthesis and its effect in evasion strategy of macrophages against Brucella infection.

Transcriptomic profiling of phospholipase A2 and the role of arachidonic acid during Brucella abortus 544 infection in both in vitro and in vivo systems.

To date, the antimicrobial activity of arachidonic acid (AA) with regard to pathogenesis of Brucella in macrophages is unknown. We found that AA is highly toxic to B. abortus in a time- and dose-dependent manner. Transcription profiling of different groups of phospholipases A2 (PLA2) was examined, ten PLA2 were detected including cPLA2-IV-A, cPLA2-IV-B, iPLA2-VI, sPLA2-I-B, sPLA2-II-C, sPLA2-II-D, sPLA2-II-E, sPLA2-V, sPLA2-X, sPLA2-XII-A. Phagocytic signaling investigation indicated that AA treatment attenuated p38α activity in infected culture macrophages possibly leading to inhibition of Brucella internalization. Post-treatment with the fatty acid did not influence bacterial intracellular multiplication or alter production of antimicrobial effectors like ROS and NO in RAW 264.7 cells. On the other hand, AA administration significantly reduced bacterial load and modestly inhibited pro-inflammatory cytokine secretion including TNF, IFN-γ and IL-6 in mice plasma. To our knowledge, we are the first to suggest that B. abortus invasion to RAW 264.7 macrophages is impaired by AA.