Macrophage Extracellular Trap Formation in Response to M. Haemolytica or its Leukptoxin is altered by Co-Incubation with Bovine Herpes Virus-1 Infected Bronchiolar Epithelial Cells

Bovine respiratory disease (BRD) is the primary cause of morbidity in the U.S. beef and dairy industry. BRD is a multifactorial disease that is caused by viral and bacterial agents leading to a severe pleuropneumonia in cattle. BRD is characterized by inflammation, intense neutrophil infiltration, consolidation and recently, extensive amounts of extracellular DNA in the lungs. One possible source of the DNA is from leukocytes that release fibrillar networks of antimicrobial protein-studded DNA matrices referred to as extracellular traps (ETs). Recently, we have demonstrated that neutrophils and macrophages produce ETs in response to Mannheimia haemolytica, an important member of the BRD complex. Previous data has demonstrated that conditioned media removed from bovine herpes virus (BHV)-1 infected bovine bronchiolar epithelial (BBE) cells contain several cytokines. Here, we examined if conditioned media from BHV-1 infected BBE cells could alter ET formation from bovine neutrophils and macrophages. We observed that bovine macrophages pre-incubated with conditioned media from BHV-1 infected BBE cells had a reduced ability to produce ETs when incubated with the leukotoxin (LKT) in comparison to the control macrophages pre-incubated with conditioned media from uninfected BBE cells. In contrast, we observed that bovine macrophages treated with conditioned media demonstrated an increase in ET formation in response to intact M. haemolytica cells. However, conditioned media-treated bovine neutrophils were unaltered in their ability to produce ETs in response to M. haemolytica or LKT. Our findings suggest that BHV infection may alter macrophage production of ETs in response to M. haemolytica or LKT, which could alter host defense

Female Neutrophils Produce Less Neutrophil Extracellular Traps in Response to the Escherichia Coli Hemolysin than Male Neutrophils

Urinary tract infections (UTIs) have been estimated to cost the U.S. approximately 1.6 billion dollars in health care costs. The majority of UTIs affect women more than men where fifty to sixty percent of women will experience a UTI in their lifetime. Uropathogenic Escherichia coli is the leading cause of UTIs. When uropathogenic E. coli is incubated in human urine, E. coli produce significantly more of the hemolysin, an RTX toxin that can lyse a wide variety of cell types. Up regulation of the hemolysin is believed to be important in E. coli colonization of bladder and kidney epithelial cells. During, a UTI there is an influx of neutrophils into the bladder, which have been demonstrated to produce neutrophil extracellular traps (NETs). NETs are composed of extracellular DNA studded with antimicrobial proteins that trap and kill various pathogens. The hypothesis of this research is that female neutrophils produce significantly less NETs in response to the E. coli hemolysin in comparison to male neutrophils. Human neutrophils and macrophages were isolated from whole blood taken from healthy male and female volunteers and were incubated for various times or with various concentrations of the E. coli hemolysin. Extracellular DNA was then quantified using PicoGreen. Red blood cells were also incubated with various concentrations of the hemolysin and red blood cell lysis was determined by the quantification of the release of heme. We observed a significant decrease in the amount of NETs produced by female neutrophils in response to the E. coli hemolysin in comparison to male neutrophils. Similarly, we observed significantly more lysis of female red blood cells as compared to male red blood cells. Preliminary results also suggest that male macrophages produce more macrophage extracellular traps (METs) than female macrophages. Our findings suggest that female neutrophils, which have a reduced ability to produce NETs and METs in response to the E. coli hemolysin, may contribute to the significant difference in the amount of UTIs women experience in comparison to men

Colonization of Methicillin-Resistant Staphylococcus Aureus in Dormitory Versus Non-Dormitory Populations

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that has developed resistance to numerous antibiotics such as methicillin, a commonly used antibiotic for the treatment of Staphylococcus infections. In the community, MRSA is widespread, and it is believed that 1% of the population is a carrier. People are potentially at a greater risk in areas where factors such as close skin-to-skin contact occur, cuts or abrasions in the skin occur, contaminated items and surfaces are present, or where overcrowded living conditions and poor hygiene are common. MRSA’s occurrence in people without risk factors who live in communities is also increasing, and some of these infections are not typically associated with staphylococci bacteria. MRSA carriage rates are important to monitor in populations where individuals are in close contact and have crowded living conditions, such as a dormitory. The goal of this research is to identify MRSA carriers in the dormitory and non-dormitory populations in order to quantify nasal carrier rates. Frequent athletic facility attendance was also monitored to determine if this is an additional risk factor. Nasal swabs samples were collected from volunteers over the age of eighteen. Background information was gathered from volunteers at the time of swabbing by means of an anonymous survey. Background information that was taken into account when analyzing data included: place of living (dormitory versus non-dormitory); if they ever lived in a dormitory and timeframe (how long and how long ago); if they had a MRSA infection previously (confirmed by a physician); how often they visited a gym facility; and their age. Our results demonstrate that approximately 10% of Winona State University student were caring MRSA intranasally. We found a strong correlation between carriage rates and dormitory status and gym usage. Similarly, dormitory populations who used a gym facility frequently were at an even higher risk of MRSA carriage. We believe these findings indicate an increase in carriage rate among WSU’s dormitory student population. These data indicate a need to increase WSU’s surveillance of MRSA carriage rates and infections

Macrophage Extracellular Trap Formation in Response to Mannheimia haemolytica or its Leukotoxin is Altered by Incubation with Conditioned Media from BHV-1 Infected Bronchiolar Epithelial Cells

Bovine respiratory disease (BRD) is a serious lower respiratory tract infection characterized by inflammation, intense neutrophil infiltration, fibrin disposition, and consolidation of the lungs. BRD, also known as shipping fever, is the primary cause of morbidity in the U.S. beef and dairy industries. It is estimated that North American loses between 600 million and 3 billion dollars per year leading to a major concern for producers. BRD is caused by various viral and bacterial pathogens including Mannheimia haemolytica and Histophilus somni. M. haemolytica, a normal nasal inhabitant, is an important bacterial agent that causes BRD. The most important virulence factor of M. haemolytica is its leukotoxin (LKT), which causes necrosis at high concentrations and apoptosis and extracellular trap (ET) formation at low concentrations. H. somni is not only another causative agent of BRD, which also causes reproductive, cardiac and neuronal diseases in cattle. It is believed that viral infection predisposes cattle to a secondary bacterial infection. Bovine herpes virus type 1 (BHV-1) is associated with BRD in cattle and has been demonstrated to synergize with M. haemolytica. Previous research has demonstrated that media removed from BHV-infected bovine bronchiolar epithelial (BBE) cells contained several cytokines including interferons, IL-1, TNF-α and IL-6. Pre-incubation of bovine neutrophils (bPMNs) with conditioned media from BHV-1 infected BBE cells increased bPMN migration, shape change, the expression of LFA-1, and the production of reactive oxygen species and degranulation. Previous research has shown that neutrophils and macrophages produce ETs in response to M. haemolytica and its LKT and H. somni. ETs are extracellular fibers of DNA and associated antimicrobial proteins that have been released by various leukocytes to trap and kill the pathogen. ET formation is distinct from apoptosis and necrosis. Here, we examine the role conditioned media, removed from BBE cells infected with BHV, plays in ET formation by bovine PMNs and macrophages in response to M. haemolytica or H. somni

Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils▿

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease

Genome divergence and increased virulence of outbreak associated Salmonella enterica subspecies enterica serovar Heidelberg

Abstract Salmonella enterica serotype Heidelberg is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing Salmonella ser. Heidelberg strains from diverse hosts and geographical regions. Human outbreak associated strains in this study were from a 2015 multistate outbreak of Salmonella ser. Heidelberg involving 15 states in the United States which originated from bull calves. Our clinicopathologic examination revealed that cases involving Salmonella ser. Heidelberg strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of Salmonella ser. Heidelberg correlated with markedly increased death losses clinically comparable to those seen in herds infected with S. Dublin, a known serious pathogen of cattle. Whole genome based single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of S. Heidelberg, is that all isolates in this cluster contained Saf fimbrial genes which are generally absent in S. Heidelberg. The acquisition of several single nucleotide polymorphisms and the gain of Saf fimbrial genes may have contributed to the increased disease severity of these Salmonella ser. Heidelberg strains