Shedding of clade 2.3.4.4 H5N8 and H5N2 highly pathogenic avian influenza viruses in peridomestic wild birds in the U.S.

European starlings (Sturnus vulgaris), house sparrows (Passer domesticus) and rock pigeons (Columba livia) are all wild birds commonly found in large numbers in and around human dwellings and domestic livestock operations. This study evaluated the susceptibility of these species to three strains of highly pathogenic avian influenza virus (HP AIV) clade 2.3.4.4 isolated in the U.S.. Experimental infection of European starlings and rock pigeons did not result in any overt signs attributable to AIV infection and no virus shedding was detected from the oral and cloacal routes. House sparrows shed by the oral route and exhibited limited mortality. Individuals from all three species seroconverted following infection. These data suggest that none of these birds are a likely potential bridge host for future HP AIV outbreaks but that their seroconversion may be a useful surveillance tool for detection of circulating H5 HP AIV.

Viral shedding of clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses by American robins.

American robins (Turdus migratorius) are commonly associated with farmsteads in the United States and have shown previous evidence of exposure to an H5 avian influenza A virus (IAV) near a poultry production facility affected by a highly pathogenic (HP) H5 virus in Iowa, USA during 2015. We experimentally infected American robins with three clade 2.3.4.4 HP H5 viruses (H5N2 and H5N8). A total of 22/24 American robins shed virus, and all three strains were represented. The highest virus titres shed were 104.3 , 104.3 and 104.8 PFU/ml, associated respectively with viruses isolated from poultry, a captive gyrfalcon (Falco rusticolus), and a Northern pintail (Anas acuta). Of those birds that shed, viral shedding was initiated 1 or 2 days post-infection (DPI) and shedding ceased in all birds by 7 DPI. This study adds an additional synanthropic wildlife species to a growing list of animals that can successfully replicate and shed IAVs.

Cottontail rabbits shed clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses.

During 2014-2015, clade 2.3.4.4 H5Nx highly pathogenic (HP) avian influenza A viruses (IAV) were first detected in North America and subsequently caused one of the largest agricultural emergencies in U.S. HISTORY: Recent evidence has suggested that cottontail rabbits can shed multiple IAV subtypes. We experimentally infected cottontail rabbits with three HP H5Nx IAVs. All rabbits tested shed virus on at least one day by at least one route. Cottontail rabbits appear to be an exception to the limited capacity for replication that has been previously reported for certain other mammalian species inoculated with clade 2.3.4.4 HP H5Nx avian influenza A viruses.

Thermoregulation of Biofilm Formation in Burkholderia pseudomallei Is Disrupted by Mutation of a Putative Diguanylate Cyclase.

Burkholderia pseudomallei, a tier 1 select agent and the etiological agent of melioidosis, transitions from soil and aquatic environments to infect a variety of vertebrate and invertebrate hosts. During the transition from an environmental saprophyte to a mammalian pathogen, B. pseudomallei encounters and responds to rapidly changing environmental conditions. Environmental sensing systems that control cellular levels of cyclic di-GMP promote pathogen survival in diverse environments. Cyclic di-GMP controls biofilm production, virulence factors, and motility in many bacteria. This study is an evaluation of cyclic di-GMP-associated genes that are predicted to metabolize and interact with cyclic di-GMP as identified from the annotated genome of B. pseudomallei 1026b. Mutants containing transposon disruptions in each of these genes were characterized for biofilm formation and motility at two temperatures that reflect conditions that the bacteria encounter in the environment and during the infection of a mammalian host. Mutants with transposon insertions in a known phosphodiesterase (cdpA) and a predicted hydrolase (Bp1026b_I2285) gene exhibited decreased motility regardless of temperature. In contrast, the phenotypes exhibited by mutants with transposon insertion mutations in a predicted diguanylate cyclase gene (Bp1026b_II2523) were strikingly influenced by temperature and were dependent on a conserved GG(D/E)EF motif. The transposon insertion mutant exhibited enhanced biofilm formation at 37°C but impaired biofilm formation at 30°C. These studies illustrate the importance of studying behaviors regulated by cyclic di-GMP under varied environmental conditions in order to better understand cyclic di-GMP signaling in bacterial pathogens.IMPORTANCE This report evaluates predicted cyclic di-GMP binding and metabolic proteins from Burkholderia pseudomallei 1026b, a tier 1 select agent and the etiologic agent of melioidosis. Transposon insertion mutants with disruptions in each of the genes encoding these predicted proteins were characterized in order to identify key components of the B. pseudomallei cyclic di-GMP-signaling network. A predicted hydrolase and a phosphodiesterase that modulate swimming motility were identified, in addition to a diguanylate cyclase that modulates biofilm formation and motility in response to temperature. These studies warrant further evaluation of the contribution of cyclic di-GMP to melioidosis in the context of pathogen acquisition from environmental reservoirs and subsequent colonization, dissemination, and persistence within the host.

Polysaccharide specific monoclonal antibodies provide passive protection against intranasal challenge with Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.

Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.

Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis.

Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS.

Substituted diphenyl ethers as a broad-spectrum platform for the development of chemotherapeutics for the treatment of tularaemia.

OBJECTIVES: The National Institute of Allergy and Infectious Disease classifies Francisella tularensis as a Category A priority pathogen. Despite the availability of drugs for treating tularaemia, the mortality in naturally acquired cases can still approach 30%. In addition, the usefulness of existing drugs for treatment in response to exposure or for prophylaxis is limited because of toxicity and delivery concerns. The aim of this study was to assess the efficacy of the lead alkyl-substituted diphenyl ether, SBPT04, in the F. tularensis murine model of infection. METHODS: SBPT04 was delivered by intraperitoneal (ip) and oral (po) routes, and mice were monitored for morbidity, mortality and relapse of disease. Pharmacokinetic studies were performed to evaluate bioavailability. Phase I and Phase II metabolism of SBPT04 was assessed in mouse and human microsomes. RESULTS: SBPT04, a potent inhibitor of the enoyl-ACP reductase enzyme ftuFabI, has efficacy against F. tularensis in the murine model of infection when delivered by both ip and po routes. SBPT04 delivered ip cleared infection by day 4 of treatment, and SBPT04 delivered po resulted in delayed dissemination. Importantly, SBPT04 delivered ip or po demonstrated efficacy with no signs of relapse of disease. Pharmacokinetic studies show increased serum concentrations following ip delivery compared with po delivery, which correlates with the observed survival rate of 100%. CONCLUSIONS: In addition to being a potent lead, this work substantiates substituted diphenyl ethers as a platform for the development of novel broad-spectrum chemotherapeutics to other bacterial agents in addition to F. tularensis.

Slow-onset inhibition of the FabI enoyl reductase from francisella tularensis: residence time and in vivo activity.

Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 microg mL(-1). The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

Fox squirrel (Sciurus niger) associations with West Nile virus.

Tree squirrels (Sciurus spp.) have been recently shown to be commonly exposed to West Nile virus (WNV). Many characteristics of WNV infections in tree squirrels are unknown. To better understand WNV associations in fox squirrels (S. niger), we conducted mark-recapture sampling (N = 72) and radio telemetry to study the longitudinal seroprevalence, seroconversions, and ectoparasites of these animals during 2005-2006 in northern Colorado. Five seroconversions were documented during this study. The majority of seroconversions occurred during the late summer/fall months. However, one seroconversion was documented over the time period of February to late March 2005. Fleas (Orchopeas howardi) were tested for WNV RNA using real-time PCR techniques. No WNV RNA positive fleas (N = 33) were detected. In addition, urine samples (N = 17) opportunistically collected from fox squirrels were negative for WNV RNA. Results indicate that seroconversions can be observed in fox squirrels during low WNV transmission years.

Antibodies to West Nile virus in feral swine from Florida, Georgia, and Texas, USA.

West Nile virus (WNV) exposure has not yet been reported in feral swine (Sus scrofa) despite the broad geographic range and population density of this species. The objectives of this study were to determine the prevalence of antibodies to WNV in feral pigs, and to evaluate serologic diagnostics as applied to this species. Feral pig serum from three states was evaluated for antibodies to WNV. The overall WNV seroprevalence rate for 222 samples collected in 2001-2004 was 22.5%. Seroprevalence rates in Florida, Georgia, and Texas were 17.2%, 26.3%, and 20.5%, respectively. The results of this study demonstrate that feral pigs could represent useful mammalian sentinels of WNV.

Antibodies to West Nile virus in asymptomatic mammals, birds, and reptiles in the Yucatan Peninsula of Mexico.

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.

Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina).

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.

Experimental transmission of West Nile virus (Flaviviridae: Flavivirus) by Carios capensis ticks from North America.

Seabird soft ticks, Carios capensis (Ixodida: Argasidae), originally collected from coastal Georgia, USA, were allowed to ingest a blood meal from pekin ducklings (Anas domesticus) infected with WNV. After 35 days of extrinsic incubation, the ticks transmitted virus to naive ducklings. WNV was detected via plaque assay and RTPCR in ticks and in tissues and serum of ducklings 7 days post infestation.

West Nile virus surveillance, Guadeloupe, 2003-2004.

We conducted extensive surveillance for West Nile virus infection in equines and chickens in Guadeloupe in 2003-2004. We showed a high seroprevalence in equines in 2003 related to biome, followed by a major decrease in virus circulation in 2004. No human or equine cases were reported during the study.

West Nile virus survey of birds and mosquitoes in the Dominican Republic.

We report West Nile virus (WNV) activity from a new area on Hispaniola, in the vicinity of Monte Cristi National Park in northwest Dominican Republic. Specific anti-WNV antibodies were detected in 12 of 58 (21%) resident birds sampled in March 2003, representing six species in the orders Cuculiformes (cuckoos), Strigiformes (owls), and Passeriformes (song birds). This seroprevalence is the highest reported from any site in the Caribbean Basin. Virus was not detected in any mosquitoes or tissues from bird specimens. Testing of 20 sick or dead birds was negative for WNV. Undetermined flavivirus antibodies were detected in four resident birds at Monte Cristi, as well as in five resident birds at Sierra de Baoruco National Park in southwest Dominican Republic. These data suggest that an unidentified flavivirus, as well as WNV, is active in the Dominican Republic.

Serologic evidence of exposure of wild mammals to flaviviruses in the central and eastern United States.

Serosurveys were conducted to obtain flavivirus and West Nile virus (WNV) seroprevalence data from mammals. Sera from 513 small- and medium-sized mammals collected during late summer and fall 2003 from Colorado, Louisiana, New York, Ohio, and Pennsylvania were screened for flavivirus-specific antibodies. Sera samples containing antibody to flaviviruses were screened for WNV-specific antibodies by epitope-blocking enzyme-linked immunosorbent assays and confirmed with plaque reduction neutralization tests. Prevalence of WNV antibodies among study sites ranged from 0% to 42.8% among the mammal communities sampled. High prevalence rates for WNV were noted among raccoons (100%, with a very small sample size, N = 2), Virginia opossums (50.0%), fox squirrels (49.1%), and eastern gray squirrels (48.3%). The high WNV antibody prevalence noted for tree squirrels, the peri-domestic tendencies of several of these species, and their ease of observation could make these species useful sentinels for monitoring WNV activity within urban communities.

Persistence of antibodies to West Nile virus in naturally infected rock pigeons (Columba livia).

Wild caught rock pigeons (Columba livia) with antibodies to West Nile virus were monitored for 15 months to determine antibody persistence and compare results of three serologic techniques. Antibodies persisted for the entire study as detected by epitope-blocking enzyme-linked immunosorbent assay and plaque reduction neutralization test. Maternal antibodies in squabs derived from seropositive birds persisted for an average of 27 days.