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Alleles from each gene were Table 1. Results Phylogenetic Characteristics of Outbreak Strains A core genome phylogenetic tree Figure 1 showed a tight cluster of the 3 Seattle cystic fibrosis outbreak strains.

The Seattle cystic fibrosis cluster was closely related to the 2 cystic fibrosis clusters described for the Papworth outbreak 6 and the Birmingham, UK, cystic fibrosis isolate 47J26 9.

Furthermore, the Seattle and Papworth cystic fibrosis outbreak strains showed some relatedness to strains CRM and GO derived strains known collectively as BRA isolated during an epidemic of soft tissue infections in Brazil 32 and the M.

The cumulative size of core segments of Mugsy alignments provides information on relatedness among groups of strains compared.

The core genome reduces in size as more genomes are added; an expected major decrease occurs after addition of more distant strains to the group.

The average genome size of cystic fibrosis outbreak strains was 4. As expected, including unrelated available clinical M.

Further addition of M. Strain GO 06 was excluded from the analysis because its genome harbors a large number of ambiguous nucleotides and an unusual hybrid appearance with fragments of M.

Strains 47J26 and M18, isolated from the sputum of a cystic fibrosis patient in Birmingham, UK, and a lymph node sample from a patient in Malaysia, respectively, were related to the outbreak strains Figure 1.

Neighbor-joining phylogenetic tree based on whole-genome multiple alignment of 24 Mycobacterium abscessus group genomes. Genomes in Table 1 were aligned by using Mugsy 22 , core segments of the alignment were identified by using Phylomark 23 , and resulting concatenated nucleotide sequences were used for construction of the midpoint-rooted neighbor-joining phylogenetic tree by using MEGA Strains from an outbreak of M.

SNPs, single-nucleotide polymorphisms. However, no information was available about any epidemiologic link between cystic fibrosis strain 47J26 to reported or unpublished outbreaks, and no clinical information was available about the patient from whom strain M18 was isolated.

Therefore, both strains were excluded from the SNP analysis. Nevertheless, SNPs for these 3 strains at positions relevant to the outbreak strains are shown in the Technical Appendix wwwnc.

A total of identical SNPs in the core segments of Mugsy alignments were shared by the 10 outbreak strains but were different in available M.

Of the SNPs, 95 gave rise to nonsynonymous mutations in several genes, including virulence factors mammalian cell entry and yrbE proteins , transcriptional regulators TetR family , and lipid metabolism genes online Technical Appendix.

Sixteen SNPs were shared only by the 3 Seattle cystic fibrosis outbreak strains, including nonsynonymous mutations in a mycobacterial large membrane protein MmpL family involved in lipid transport and virulence 34 and genes involved in amino acid and energy metabolism Figure 2; online Technical Appendix.

Eighty-six SNPs were present only in strain CRM soft tissue outbreak from Brazil, including nonsynonymous mutations in an MmpL family protein; transcriptional regulators; and lipid, amino acid, and energy metabolism genes Figure 2; online Technical Appendix.

Alignment of the MmpL family protein with distinct MmpL proteins described above for the Seattle cystic fibrosis outbreak and the Brazil soft tissue outbreak showed diversity at several amino acid residues in all 3 proteins.

We also searched for polymorphisms associated with macrolide and aminoglycoside resistance. The Papworth Figure 2. Venn diagram of core single-nucleotide polymorphisms SNPs shared by outbreak localities.

Core segments of the Mugsy 22 alignment of the 20 Mycobacterium abscessus subsp. Strains 19f, 14h, 12c, and 28a, representative of Papworth cluster 1, and Seattle strains shared the AG mutations in 16S rRNA, which conferred aminoglycoside resistance In the first approach, we retrieved rpoB sequences from the 6 genomes of representative strains of the Papworth cystic fibrosis outbreak and performed partial sequencing of the rpoB gene for selected isolates from the Seattle cystic fibrosis outbreak.

We then compared these sequences with those of isolates from the outbreak in Brazil and unrelated clinical isolates comprising M.

However, none of the M. Most of the 26 M. However, 4 strains harbored this signature Table 2 29, Multiple alignment of rpoB sequences among available M.

Multiple alignment of secA1 sequences among available M. Further analysis of secA1 sequences from 12 M. Those 2 strains were included among the 4 strains that had the 2-SNP rpoB signature.

We also developed a simple MLST protocol that could be used as a second confirmatory assay. Alleles for each of 13 housekeeping genes cya, gdhA, argH, glpK, gnd, murC, pgm, pknA, pta, pur, rpoB, hsp65, and secA1 were extracted and concatenated for each M.

The Seattle and Papworth cystic fibrosis outbreak strains grouped together in the tree with cystic fibrosis strain 47J26 and isolate M18 from Malaysia Figure 3.

Thus, partial sequencing of rpoB and secA1 gens, followed by target MLST analysis, could be used to rule out isolates as belonging to these 2 cystic fibrosis clusters.

Neighbor-joining phylogenetic tree based on target multilocus sequences types from 20 Mycobacterium abscessus subsp.

Electronic PCR was performed on the M. Nucleotide sequences from each gene were concatenated for each genome and aligned by using ClustalW 31 , and the core alignment was used for construction of a midpoint-rooted neighbor-joining phylogenetic tree by using MEGA The longer branch length for Papworth isolate 12c was caused by low-quality nucleotides single-nucleotide polymorphisms [SNPs] located at the edge of Velvet contigs.

Discussion The implications of this study are extensive. Currently, most experts recommend identifying isolates of M.

This report further corroborates these recommendations and places even greater pressure on clinical laboratories to fully identify M. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness 4,, nt core genome alignment size, 11 shared unique SNPs with each other and major-level relatedness 4,, nt core genome alignment size with soft tissue epidemic strains from Brazil.

Genomic features shared between strains from all 3 outbreaks might make them more transmissible, whether from patient to patient directly or indirectly as in cystic fibrosis outbreaks or from a common source, as in soft tissue infections.

We speculate that some of these specific genomic traits may be favorable for the successful establishment of epidemic soft tissue infections.

A previous study did not detect a common source or person-to-person transmission of the M. Our findings emphasize the necessity of screening all isolates of M.

Because of evidence supporting patient-to-patient transmission of multiple different respiratory tract organisms, the Infection Control Guidelines currently in draft form for public comment of the United States Cystic Fibrosis Foundation CFF www.

Patients with cystic fibrosis are advised not to attend indoor meetings with other cystic fibrosis patients CFF and Infection Prevention and Control Guidelines It remains unclear why intercontinental organisms are so closely related.

One hypothesis is that direct patient contact led to transmission. The Seattle index case-patient traveled to British Columbia, Canada, before and after acquiring mycobacterial infection, to Oregon before mycobacterial infection, and to Atlanta, Georgia, and Bethesda, Maryland, after mycobacterial infection.

However, the patient did not report any contact with other cystic fibrosis patients at these destinations.

A second hypothesis is that the mycobacterial strain could have been carried by persons with cystic fibrosis who were clinically well.

A third hypothesis is that there was an independent selection of M. Availability of additional whole-genome sequencing data tracking the global epidemiology of the M.

In addition, this data will help delineate global clusters of M. Addendum Recent whole-genome data show deep genetic separation of 3 subspecies, ruling against grouping M.

Acknowledgments We thank Josephine Bryant, Dorothy Grogono, Julian Parkhill, and Andres Floto for their help and for providing sample identification and accession numbers for the Papworth outbreak isolates.

H, and M. Carter Foundation. His primary research interests are the use of comparative and functional genomics to understand bacterial diversity and virulence, study host-pathogen interactions, and identify vaccine candidates and drug targets to cure disease.

Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel.

Nontuberculous mycobacteria. I: multicenter prevalence study in cystic fibrosis. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France.

Mycobacterium abscessus and children with cystic fibrosis. Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center.

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.

Genome sequence of an epidemic isolate of Mycobacterium abscessus subsp. Genome Announc. Complete genome sequence of Mycobacterium massiliense.

J Bacteriol. Whole-genome sequence of the emerging pathogen Mycobacterium abscessus strain 47J Genome sequence of Mycobacterium massiliense M18, isolated from a lymph node biopsy specimen.

Genomic analysis of Mycobacterium massiliense strain M, an isolate from human sputum. Genomic analysis of Mycobacterium abscessus strain M, which has an ambiguous subspecies taxonomic position.

Annotated genome sequence of Mycobacterium massiliense strain M, belonging to the recently created taxon Mycobacterium abscessus subsp.

Complete genome sequence of Mycobacterium massiliense clinical strain Asan , belonging to the type II genotype.

Genomic insights into the emerging human pathogen Mycobacterium massiliense. Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus.

Mol Microbiol. Sep 3 [Epub ahead of print]. Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.

Draft genome sequence of Mycobacterium abscessus subsp. Draft genome sequence of Mycobacterium bolletii strain M24, a rapidly growing mycobacterium of contentious taxonomic status.

Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. Mugsy: fast multiple alignment of closely related whole genomes.

Phylomark, a tool to identify conserved phylogenetic markers from whole-genome alignments. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.

Mol Biol Evol. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Inaccuracy of single-target sequencing for discriminating species of the Mycobacterium abscessus group.

Cohort study of molecular identification and typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii.

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus sensu lato strains. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

Nucleic Acids Res. Epidemic of postsurgical infections caused by Mycobacterium massiliense. Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.

Future Microbiol. MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria. Chem Biol. The detection and sequencing of a broadhost-range conjugative IncP-1beta plasmid in an epidemic strain of Mycobacterium abscessus subsp.

Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus.

Antimicrob Agents Chemother. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae.

J Infect Dis. New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identification of Mycobacterium massiliense with inducible clarithromycin resistance.

Clinical significance of differentiation of Mycobacterium massili ense from Mycobacterium abscessus. Lack of transmission of Mycobacterium abscessus among patients with cystic fibrosis attending a single clinic.

Heine, Glenn A. Marsh, Christopher C. Broder, and Lin-Fa Wang In recent years, the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health.

For example, Hendra virus HeV , a zoonotic paramyxovirus, was discovered in , and since then, infections have occurred in 7 humans, each of whom had a strong epidemiologic link to similarly affected horses.

As a consequence of these outbreaks, eradication of bat populations was discussed, despite their crucial environmental roles in pollination and reduction of the insect population.

We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse, human, and environmental health.

The HeV vaccine for horses is a key example of a One Health approach to the control of human disease. H endra virus HeV is an emerging zoonotic paramyxovirus for which natural reservoirs are the 4 species of flying fox Pteropus bats found on mainland Australia 1.

Middleton, J. Pallister, R. Klein, J. Haining, R. Arkinstall, L. Frazer, J. Bingham, D. Johnson, J. White, A. Foord, H.

Heine, G. Marsh, L. Feng, C. Huang, N. Edwards, M. Wareing, M. Elhay, Z. Each casepatient had a strong epidemiologic connection to similarly affected horses through exposure to equine secretions late in the incubation period, during terminal illness, or at the time of postmortem examination of infected animals 2 : no human case of HeV infection has been attributable to direct spillover from bats 3.

There is no licensed anti-HeV therapeutic drug for use in any species. However, gene copy numbers increased exponentially with the onset of fever, when viral genome could also be recovered from blood, oral secretions, urine, and feces 6.

Rapid progression of clinical signs, as observed in equine field cases of this disease, led to euthanasia of experimental animals on humane grounds.

Viral RNA was recovered from all tissues sampled at postmortem examination, and virus was reisolated from lung, brain, lymphoid tissues, and kidney 6.

In accordance with epidemiologic observations 2 , it was concluded that HeV-infected horses in the immediate presymptomatic or symptomatic stages of disease pose a high risk for transmission of HeV to humans.

This risk is then exacerbated because it is symptomatic horses that come to the attention of veterinarians, leading to various 1 These authors contributed equally to this article.

One of the sides of each pen was able to be moved in toward the horse on a ratchet mechanism, allowing staff close access to the horses, as required, over the side of the pen without the need for them to enter the pen itself Horses were fed a mixture of lucerne alfalfa and grass hay, concentrates, and specified fruit and vegetables.

On the day before HeV exposure, an indwelling jugular catheter was sutured in position, and an intrauterine temperature data-logger was placed into each horse.

The humane end point was defined as fever for up to 48 h accompanied by increased respiratory rate, dyspnea, depression, ataxia, or pressing the head against the side of the stall.

Euthanasia was conducted by intravenous injection of a barbiturate following sedation with intravenous detomidine and butorphanol. Ferrets and guinea pigs used as controls in efficacy studies to confirm pathogenicity of the inoculum were housed in pairs in the BSL-4 facility, given species-appropriate dry rations and dietary treats, and provided with water ad libitum.

While in the BSL-4 animal room, staff wore fully encapsulated suits with an external air supply.

These events were accompanied by a marked rise in the number of HeV-related media reports. The reports had an increasingly politicized focus on the role and control of flying foxes as carriers of HeV 8 and a deemphasis of the critical role played by horses in HeV transmission to humans.

Heightened public awareness of the risk that infected horses posed to humans persisted and was paralleled by increased numbers of veterinarians leaving equine practice because of personal safety and liability concerns 9.

The considerable investment in education and improved infection control measures that had been implemented did not effectively mitigate perceptions around the risks associated with the routine veterinary care of horses The actual mechanism of HeV transmission from bats to horses is probably complex and dependent upon socioeconomic, environmental, and ecologic factors 11 , and there is currently no straightforward solution for preventing transmission.

Eradication of flying foxes would pose extraordinary operational challenges, notwithstanding attendant moral, ethical, and environmental issues, and eliminating the interface between bats and horses is impractical for periurban and rural communities.

The most direct approach for reducing the risk posed to humans by HeV-infected horses would be implementation of a strategy that will lead to suppression of virus replication in horses.

We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse and human health.

The emergence of several highly pathogenic zoonotic diseases in humans in recent years has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health.

All subsequent vaccines were formulated with clarified CHO cell culture supernatant that was then gamma irradiated.

The change of the expression system from F cells to CHO cells was driven by the need for higher antigen yields, and equivalence was supported by laboratory analysis of the expressed antigens from the 2 systems and a comparison study in ferrets.

Vaccine formulations used in efficacy studies are summarized in Table 1. Immunization All immunizations comprised two 1-mL doses administered intramuscularly 3 weeks apart, unless stated otherwise.

Overall, 4 efficacy tests were completed; 2 vaccinated horses were used in the first test, 3 were used in the second, 2 were used in the third, and 3 were used in the fourth.

For the 4 tests, a pathogenicity control for the inoculum was provided by 1 horse test 1 , 4 guinea pigs test 2 , 2 ferrets test 3 , and 2 ferrets test 4.

Exposure conditions for 3 additional unvaccinated control horses were equivalent to those used in both vaccinated horses and the inoculum-control horse and have been described 6.

Sample Collection and Analysis During efficacy studies, nasal, oral, and rectal swab samples; urine and feces samples; and blood samples in EDTA were collected from the horses before virus exposure and then daily until the animals were euthanized.

At postmortem examination, the following tissues were collected for viral genome detection, virus isolation, histopathology, and immunohistochemistry according to 15 : adrenal gland, bladder, brain including olfactory pole , cerebrospinal fluid, guttural pouch, heart, kidney, large intestine, liver, lung, lymph nodes bronchial, inguinal, intermandibular, mandibular, renal , meninges, nasal turbinates, ovaries, pharynx, small intestine, spinal cord, spleen, sympathetic nerve, trigeminal ganglion, and uterus.

The following analyses were conducted as described 15 : quantitative reverse transcription PCR for the detection of the HeV N gene, histology, immunohistology, serum neutralization test, and virus isolation.

Table 1. Infection characteristics for 3 of these unvaccinated animals have been described 6 ; data from the fourth control animal was gathered as part of the current work.

In that fourth control, onset of fever accompanied by a rising heart rate was noted on postchallenge day 6. On postchallenge day 7, the horse became clinically depressed, its temperature and heart rate continued to rise, and it was euthanized.

Gross postmortem findings included pleural thickening and moderate dilation of the lymphatic vessels on the ventral 10 cm of the cardiac lung lobes.

Histologic examination revealed systemic vasculitis affecting the lung Figure 1, panel A , spleen, kidney, nasal epithelium, lymph nodes, and brain; alveolitis; and lymphadenitis.

HeV antigen was identified in endothelial cells and vascular walls within lung, brain Figure 1, panel B , nasal epithelium, lymph nodes, spleen, kidney, liver, myocardium, salivary gland, pharynx, small intestine, uterus, ovary, and adrenal gland, as well as in myocardial fibers and glomeruli.

Viral RNA from this fourth control horse was detected in nasal swabs collected on postchallenge day 3 Table 2; summarized in Table 3 and also in blood collected immediately before the onset of fever.

After onset of fever, but before development of other clinical signs of illness, HeV RNA was also detected in the oral swab sample.

On the day of euthanasia, genome was detected in oral and nasal swab samples, blood, rectal swab, and urine samples; however, virus was not reisolated from any sample collected before postmortem examination.

Viral RNA was detected in all tissues sampled at postmortem examination except cerebrospinal fluid. Reisolation of virus was attempted for all tissues: HeV was recovered from lung, submandibular lymph node, small intestine, large intestine, and adrenal gland.

In a series of vaccine efficacy studies, 10 horses were immunized with HeVsG glycoprotein and then exposed to an otherwise lethal dose of HeV by the oronasal route.

Each study also included a pathogenicity control for the virus inoculum. In the first of these, the pathogenicity control was the fourth control horse described above.

Together with historical data gathered from 3 horses following their exposure to HeV under equivalent experimental conditions 5 , data from this horse completed the requirements of the Australian Pesticides and Veterinary Medicines Authority for defining the horse infection model.

In subsequent studies, guinea pigs or ferrets were used as pathogenicity controls to maximize the number of vaccinated horses that could be accommodated in the BSL-4 facility.

These animals duly displayed signs, lesions, tissue antigen and viral genome distribution, and virus reisolation data consistent with acute HeV infection.

In contrast to unvaccinated control horses, vaccinated horses remained clinically healthy during the observation period after exposure to HeV.

Following elective euthanasia at the time of predicted peak viral replication, there was no gross or histologic evidence of HeV infection in vaccinated horses; all tissues examined were negative for viral antigen by immunohistochemistry; and viral genome was not recovered from any tissue, including nasal turbinates, pharynx, and guttural pouch Table 3.

For 9 of 10 vaccinated horses, viral RNA was not detected in daily nasal, oral, or rectal swab specimens or from blood, urine, or feces samples collected before euthanasia, and virus was not reisolated from any of these clinical samples.

Virus was not reisolated from these samples. At the time of euthanasia, no rise in antibody titer was detected in any vaccinated horse following exposure to HeV.

Figure 1. A Hematoxylin and eosin staining shows systemic vasculitis affecting the lung. B Immunohistologic examination, using polyclonal rabbit antiNipah N protein, indicates Hendra virus antigen in a blood vessel in the brain.

Cyclethreshold values were converted to relative copy numbers by using a standard curve of a sample with a known copy number.

Discussion The formal launch of the HeV horse vaccine in November represents the culmination of multiple studies conducted in several animal infection models over the course of many years.

Where evidence of low-level virus replication did occur in secretions, it was transient and unaccompanied by the development of clinical illness, and virus was not isolated from the secretions.

The henipavirus surface-expressed G glycoprotein has the critical role of initiating infection by binding to receptors on host cells, and antibodies directed against this protein can neutralize virus Thus it is likely that, as seen for other paramyxoviruses with a viremic infection phase e.

Similar results were obtained for 2 of 3 horses exposed to HeV 6 months after vaccination. In the third horse, which also remained clinically healthy, evidence of HeV replication was limited to low-level transient detection of viral genome but not virus from the nasal cavity.

In assessing the field significance of this observation, the following must be noted: the experimental horses were exposed to considerably higher levels of HeV than have been recovered from flying foxes 1 , higher levels of viral genome were routinely found in the nasal secretions of nonimmunized horses, and all human infections have been acquired from animals in which clinical disease developed.

ID, identification;dpc,daysafterchallenge;PM,postmortem. In previous henipavirus vaccine efficacy studies in cats and ferrets, a neutralizing antibody titer of 32 was shown to be protective against the development of clinical disease In the horse efficacy studies, the 3 horses with prechallenge antibody titers of 16 or 32 were similarly protected from clinical illness.

However, we caution that any correlation between antibody titer at the time of exposure to virus and levels of subsequent protection against infection and disease is unlikely to be linear; it is possible that animals with even lower titers will have epidemiologically meaningful protection against HeV exposure occurring in the field, not least because of stimulation of immunological memory.

Additional studies assessing the duration of protection are planned, and the outcome of these will further inform recommendations regarding booster vaccination.

In other regions where HeV infection of horses has not been reported, there is understandably more uncertainty regarding the value of vaccination as part of horse preventative health programs.

Any reluctance to vaccinate horses against HeV that is based on assessment of risk is probably exacerbated by several factors, including the novelty of the vaccine roll-out process to the Australian horse industry, a mistaken perception that fast-tracking vaccine release involved overlooking key safety and efficacy issues, the lack of published data on safety in pregnant mares, reluctance of certain industry sectors to vaccinate because of import restrictions on HeV-seropositive horses, and cost.

Although it is likely that each of these barriers will diminish over time, our experiences may assist the development of road maps to guide the future release of vaccines against BSL-4 pathogens that are associated with highly sporadic disease events and where the decision to vaccinate is in the hands of the persons whom vaccination was designed to protect.

Several recently emerged zoonotic viruses, including HeV, Nipah, Ebola, and Marburg viruses, are classified as BSL-4 agents because of their ability to cause severe illness or death in humans and because there have been no effective vaccines or postexposure treatments to protect against the diseases they cause.

The vaccine against HeV Equivac HeV is a commercially deployed vaccine developed against a BSL-4 agent and is the only licensed treatment for henipavirus infection.

Development of vaccines against BSL-4 agents for use in humans requires that the US Food and Drug Administration implement the animal rule, which requires that such vaccines first be tested for efficacy in at least 2 animal models As a veterinary vaccine, Equivac Figure 2.

Days represent days after challenge. At the same time, the vaccine is expected to provide a substantial health benefit to humans.

In so doing, this vaccine encapsulates the spirit of a One Health approach, not just in terms of the interconnectedness of human and animal health but also with respect to environmental health.

One consequence of the recent HeV outbreaks was a move to eradicate bat populations, despite their crucial environmental roles in pollination and reduction of the insect population.

Successful deployment of the HeV vaccine, with a targeted reduction in the risk for acute disease events in horses and humans, should help reduce the current momentum toward the setting of control policies with potential adverse effects on the environment.

The current HeVsG glycoprotein vaccine technology provides a platform for the rapid development of related vaccines to counter future emergent threats.

Dr Middleton, a veterinarian with a PhD in pathology, works as a senior principal research scientist. Her research interest is the pathogenesis of emerging infectious diseases including highly pathogenic avian influenza viruses, henipaviruses, severe acute respiratory syndrome, and bat-borne viruses in reservoir and spillover hosts.

Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission.

Am J Trop Med Hyg. Human Hendra virus encephalitis associated with equine outbreak, Australia, Ecological aspects of Hendra virus.

Curr Top Microbiol Immunol. Transmission studies of Hendra virus equine morbillivirus in fruit bats, horses and cats. Aust Vet J.

Animal models of henipavirus infection. Vet J. Henipaviruses in their natural hosts. Degeling C, Kerridge I. Hendra in the news: public policy meets public morality in times of zoonotic uncertainty.

Soc Sci Med. Unexpected result of Hendra virus outbreaks for veterinarians, Queensland, Australia.

Hendra virus: an emerging paramyxovirus in Australia. Lancet Infect Dis. Global trends in emerging infectious diseases.

PLoS Med. Animal experimentation in level 4 facilities. In: Richmond JY, editor. Anthology of biosafety: BSL-4 laboratories.

Receptor binding, fusion inhibition and induction of cross-reactive neutralizing antibodies by a soluble G glycoprotein of Hendra virus.

J Virol. Feline model of acute Nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine. A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats.

A Hendra virus G glycoprotein subunit vaccine protects African green monkeys from Nipah virus challenge. Sci Transl Med.

Henipavirus mediated membrane fusion, virus entry and targeted therapeutics. Immunization strategies against henipaviruses.

Berlin: Springer; A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute Nipah virus infection.

PLoS Pathog. A neutralizing human monoclonal antibody protects African green monkeys from Hendra virus challenge.

Nipah virus: vaccination and passive protection studies in a hamster model. Antibody prophylaxis and therapy against Nipah virus infection in hamsters.

Acute Hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model. Immunization against viral diseases.

Martin MA, et al. Fields virology. Protective effects of glycoprotein-specific monoclonal antibodies on the course of experimental mumps virus meningoencephalitis.

Plotkin SA. Vaccination against the major infectious diseases. Prevalence of henipavirus and rubulavirus antibodies in pteropid bats, Papua New Guinea.

Henipavirus RNA in African bats. Henipavirus infection in fruit bats Pteropus giganteus , India. Mycobacterium abscessus was first isolated from gluteal abscesses in a year-old patient who had injured her knee as a child and had a disseminated infection 48 years later.

The species M. In current taxonomy, M. Sources 1. Int J Syst Evol Microbiol. Proposal that Mycobacterium massiliense and Mycobacterium bolletii be united and reclassified as Mycobacterium abscessus subsp.

An unusual acid-fast infection of the knee with subcutaneous, abscess-like lesions of the gluteal region.

J Invest Dermatol. Jones, Stephanie Sonnberg, Zeynep A. Webby, and Robert G. Several subtype H7N9 isolates contain influenza genes previously identified in viruses from finch-like birds.

Because wild and domestic songbirds interact with humans and poultry, we investigated the susceptibility and transmissibility of subtype H7N9 in these species.

Finches, sparrows, and parakeets supported replication of a human subtype H7N9 isolate, shed high titers through the oropharyngeal route, and showed few disease signs.

Virus was shed into water troughs, and several contact animals seroconverted, although they shed little virus. Our study demonstrates that a human isolate can replicate in and be shed by such songbirds and parakeets into their environment.

T he emergence of novel influenza strains from the avian reservoir remains a constant threat to human and animal health, as was recently illustrated by human infections with novel and wholly avian influenza A H7N9 viruses in China.

These viruses show little virulence in birds but can cause severe illness in humans 1,2. Jones, S. Sonnberg, Z. Kocer, K. Shanmuganatham, P.

Seiler, R. Webby, R. Zhu, Y. Guan, M. In the 3 index case-patients, the illness progressed to acute respiratory distress syndrome and death 1 , and most persons with confirmed infections required hospital care 2,5.

Several of the A H7N9 virus internal genes polymerase basic protein [PB] 1, matrix, nonstructural protein, and nucleoprotein originated from the H9N2 subtype commonly found in chickens.

When chickens and quail were inoculated with A H7N9 isolated from humans, they shed the viruses to high titers but had little or no clinical disease 9, Thus, poultry appears to be a reservoir for A H7N9 viruses and a source of human infections.

Therefore, songbirds and other small, terrestrial birds could have been directly involved in the genesis of novel A H7N9 viruses and subsequent infection in humans.

Songbirds are common household pets and are in close contact with humans and domesticated animals.

Their wild counterparts also are likely to interact with poultry in backyard farms and in many farming sectors 14, For this study, we chose 3 species of Passeriformes zebra finches, society finches, and sparrows , which are related to the bramblings described previously.

The study was conducted during June and July at St. Pooled allantoic fluid was used for each study. We serologically tested 3 or 4 sentinel birds of each species excluding sparrows because of limited availability for influenza antibodies H3, H5, H7 by hemagglutination inhibition HI assay and found them to be antibody negative.

Swabs taken on day 0 were negative for virus isolation in eggs. Food was provided ad libitum, and a minimum of 0.

All birds within a given group shared the same water and food troughs. All animal experiments were approved by the St.

Necropsy At 3 days post inoculation dpi , 2 finches from each group and 1 sparrow were euthanized for necropsy. Parakeets were excluded from euthanasia and subsequent necropsy because of limited numbers.

To prevent cross-contamination, organs were harvested in the following order, and instruments were cleaned after each organ was sampled: brain, eye, lung, trachea, small intestine, and large intestine.

Tissues were homogenized, and virus was isolated and titrated in eggs as described 15, Birds that were found dead underwent similar necropsy, but only brain, lung, and combined small and large intestinal tissue were collected 15,19, Results Replication and Pathogenicity of A H7N9 Virus All inoculated birds shed virus, but shedding was confined to the oropharynx; no virus was isolated at any time from the cloaca.

Shedding was highest in the 2 finch species at 2 dpi, and virus titers shed by these birds were 1. Virus had cleared in all inoculated animals by 8 dpi.

One sparrow and 1 zebra finch were found dead at 3 and 6 dpi, respectively Table 1 , but only the sparrow had shown clinical signs of disease lethargy; loose, discolored feces; ruffled feathers.

All cloacal samples were below the limit of detection at all time points. Shedding of Virus into Water Each day for 6 days, water was sampled from the communal trough shared by birds within each cage group, and virus was titrated in eggs.

Virus was detected in all water troughs and on multiple days Figure. Both finch species shed virus into the water on every postinoculation day studied, with the exception of 3 dpi in the zebra finches.

Virus was not shed into the water until 3 dpi by the sparrows and parakeets. However, the possibility that bird groups consumed different levels of water on any given day could not be normalized.

A single contact zebra finch showed trace amounts of virus at 2 and 4 dpi, and 2 sparrows showed trace amounts of virus at 4 dpi.

Contact parakeets remained virus negative. In contrast, 1 contact society finch shed high titers of virus from 2 dpi As with the inoculated animals, direct contacts shed virus only by the oropharyngeal route.

Isolation of Virus from Organs Organs from inoculated birds were recovered 3 dpi, and virus was isolated and titered in chicken eggs.

The sparrow that underwent necropsy showed trace virus only in the lungs Table 3. Both finch species showed high virus titers in the trachea 4.

In the zebra finches, virus was observed only in the tracheas, consistent with swab findings, but 1 of 2 society finches showed trace amounts of virus in the brain and eye, whereas the other had trace amounts in the small and large intestine and high lung virus titer 5.

Two donor birds one sparrow and one zebra finch died during the experiment and underwent necropsy. However, in the zebra finch, virus was detected in brain, lung, and intestine 2.

Rates of Seroconversion Figure. Virus shedding into water trough. The lower limit of detection was 0.

Mean HI titers in inoculated birds ranged from 4. All contact zebra finches seroconverted, but only 1 of 3 society finches and 2 of 3 sparrows seroconverted.

No seroconversion of contact parakeets was observed. Mean HI titers in contact animals that seroconverted were 4. Mean titers were highest in society finches and lowest in parakeets, although they did not differ significantly in inoculated versus contact groups or according to species.

HI titers to heterologous human subtype H5 and songbird subtype H3 viruses were negative Table 4. Shedding was limited to the oropharynx, which may have reduced contact transmission; however, at most times sampled, the water troughs contained large amounts of virus.

Furthermore, low-pathogenicity influenza viruses those lacking a multibasic cleavage site in the hemagglutinin protein , such as the A H7N9 isolate used here, remain stable in water longer than their highly pathogenic counterparts 22 and could therefore serve as an inoculum for cage mates.

Despite little shedding in the contacts, seroconversion of at least 1 contact animal in each of the finch and sparrow groups indicates exposure with antigenic epitopes of the subtype H7 hemagglutinin.

The parakeets in particular showed no contact animal shedding or seroconversion, a phenomenon we have previously observed with an A H3N8 isolate from a songbird R.

Webster et al. This observation may have implications for risk assessment of this species in the pet bird trade; however, the lack of proper influenza transmission data with this species in the literature warrants additional studies to confirm the validity of this observation.

One direct-contact society finch shed virus equivalent to titers in inoculated birds and shed for a longer period, which suggests that efficient transmission and replication in contact animals, although rare, is possible.

The ability of these small birds to harbor and shed A H7N9 viruses, usually with few signs of illness, creates a substantial potential for transmission to humans, as well as to poultry and wild birds.

Interspecies transmission has not yet been investigated, and the extent to which A H7N9 -infected finches, sparrows, and parakeets may transmit virus to other species, including through shared water sources, is unknown.

Two host groups have the greatest potential interaction with small birds. The first is domesticated poultry, primarily chickens but also a wide variety of gallinaceous and game birds.

The peridomestic nature of songbirds facilitates an interaction with poultry in large production facilities and in backyard farms 14, In these cases, they may share common food and water sources The interspecies transmission of influenza from songbirds to poultry is not without precedent.

Nestorowicz et al. Forrest et al. In the case of the low-virulence A H7N9 viruses, most inoculated birds shed virus while remaining clinically healthy.

The absence of illness and death in A H7N9 -infected birds has implications for a greater quantity and duration of virus shedding into the environment, as well as higher activity levels and likelihood of interaction with other susceptible hosts.

Preliminary data suggest that chickens and quail are highly susceptible to infection with this human A H7N9 isolate and that the virus is readily transmitted by direct contact 9, The high titers recovered from the shared water troughs of all species tested in our study suggest that the virus could be transmitted to poultry and other birds through this route.

Therefore, reduced interaction of domestic poultry with wild passerine birds is advisable, although this precaution might not be feasible in developing countries where numerous backyard farms lack biosecurity.

Comprehensive biosecurity also is often lacking at large poultry farms in Table3. A second host group with high potential to interact with songbirds and other small terrestrial birds are humans.

Finches, sparrows, and parakeets are not only common in the wild but are popular pets worldwide They are often sold in the live-bird markets of eastern Asia, where the risk for zoonotic influenza transmission of H7N9 and other influenza subtypes is already established 26, Such pet birds may be procured from the wild and may have been exposed to a variety of pathogens before entering the market chain 9.

In China, the keeping of pet birds is associated with luck 28 and is common among elderly men, who often stroll through the parks with their caged birds 28, In caring for such pets, their owners could become infected by virus contaminated drinking water or from fomites on the feathers deposited while bathing in water troughs or from saliva while preening This same demographic group elderly men experienced disproportionate rates of illness and death from A H7N9 infection in China A recent epidemiologic study by Rivers et al.

Although completely avoiding contact with pet birds during an avian influenza outbreak might not be feasible, surveillance of such species in the markets, and perhaps in the wild, would help to identify or rule out previously unsuspected hosts that might support or disseminate emerging viruses.

To expand on our findings, future studies should include the examination of genetic changes in the human A H7N9 virus during replication and transmission in the songbirds and parakeets.

Sequence analysis of virus shed by the small birds in our study might indicate which species would be most receptive to transmission.

The loss of the markers of mammalian adaptation and reversion to an avian-like genotype might predispose the virus to transmission from the small birds to poultry and other birds, rather than to humans.

Additionally, studies that assess the sharing of housing, water, and food by inoculated songbirds and poultry particularly chickens or quail would shed light on the interspecies transmission potential of A H7N9 viruses.

Our demonstratration that parakeets and multiple species of songbirds are susceptible to influenza A H7N9 virus isolated from humans during the recent outbreak in China further supports the possible contribution of songbirds and parakeets to the ecology, maintenance, and transmission of novel A H7N9 viruses.

Finally, they lead us to propose that finches, sparrows, and parakeets may be intermediate hosts and sources of A H7N9 viruses and that their frequent interaction with wild birds, domestic poultry, and humans renders them a particular risk factor in the emergence and transmission of novel influenza strains.

This work was supported by contract no. Dr Jones is a postdoctoral fellow working at St. Human infection with a novel avian-origin influenza A H7N9 virus.

Human infection with avian influenza A H7N9 virus: an assessment of clinical severity. Geneva: The Organization; Exposure to avian influenza H7N9 in farms and wet markets.

Emerging influenza. J Clin Virol. The novel H7N9 influenza a virus: its present impact and indeterminate future.

Vector Borne Zoonotic Dis. Branswell H. Chicken, quail catch and shed high volumes of new H7N9 flu, study shows. The Canadian Press. Toronto: Shaw Media; [cited Aug 16].

Euro Surveill. Rapid reassortment of internal genes in avian influenza A H7N9 virus. Infectious and lethal doses of H5N1 highly pathogenic avian influenza virus for house sparrows Passer domesticus and rock pigeons Columbia livia.

J Vet Diagn Invest. Virus shedding and potential for interspecies waterborne transmission of highly pathogenic H5N1 influenza virus in sparrows and chickens.

Vet Pathol. Arch Virol. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. Am J Hyg. Role of terrestrial wild birds in ecology of influenza A virus H5N1.

Hemagglutination inhibition test. Advanced laboratory techniques for influenza diagnosis.

Atlanta: US Department of Health. Education, and Welfare; Persistence of H5 and H7 avian influenza viruses in water. Avian Dis.

Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: role of passerine birds in maintenance or transmission?

Shedding and serologic responses following primary and secondary inoculation of house sparrows Passer domesticus and European starlings Sturnus vulgaris with low-pathogenicity avian influenza virus.

Avian Pathol. Eurasian tree sparrows, risk for H5N1 virus spread and human contamination through Buddhist ritual: an experimental approach.

Zoonoses in pet birds: review and perspectives. Vet Res. Estimating human cases of avian influenza A H7N9 from poultry exposure.

Driedger M. Cheng J. Can preening contribute to influenza A virus infection in wild waterbirds? Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases.

Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses.

Webster, Department of Infectious Diseases, St. Amman, David Wong, Craig R. Manning, Stuart T. Nichol, Pierre E.

Rollin, Dongxiang Xia, James P. Watt, and Duc J. An investigation encompassing clinical, epidemiologic, laboratory, and environmental factors identified 10 cases among residents of 3 states.

Eight case-patients experienced hantavirus pulmonary syndrome, of whom 5 required intensive care with ventilatory support and 3 died.

Rodent nests and tunnels were observed in the foam insulation of the cabin walls. All signature tent cabins were closed and subsequently dismantled.

Continuous public awareness and rodent control and exclusion are key measures in minimizing the risk for hantavirus infection in areas inhabited by deer mice.

H antavirus pulmonary syndrome HPS is an acute viral disease, characterized by a nonspecific febrile illness followed by severe noncardiogenic pulmonary edema and cardiogenic shock.

It is also referred to as hantavirus cardiopulmonary syndrome. Fritz, B. Enge, M. Novak, V. Kramer, S. Messenger, M.

Niemela, D. Xia, J. Watt, D. Knust, L. Osadebe, C. Amman, C. Manning, S. Nichol, P. Buttke, D.

Deer mice Peromyscus maniculatus are the reservoir for SNV 8 , which infected mice shed in their urine, saliva, and feces.

Humans are exposed chiefly by inhaling aerosolized excreta. Person-to-person transmission of SNV has not been documented Although household clusters of HPS have been reported 13,14 , most HPS cases occur as single, sporadic disease incidents.

In August , the California Department of Public Health CDPH confirmed HPS in 2 California residents, both of whom had visited Yosemite National Park Yosemite in June and had lodged in so-called signature tent cabins which differ from regular tent cabins by having an interior wall and roof consisting of drywall with a layer of foam insulation between the drywall and exterior canvas in the Curry Village area of Yosemite Valley.

This report summarizes the clinical, epidemiologic, laboratory, and environmental findings of the investigation.

Members of the Yosemite Hantavirus Outbreak Investigation Team are listed at the end of this article. Finding case-patients was facilitated through health alerts issued by CDPH, NPS, and CDC to health care providers, advising them to contact state or local public health officials to obtain confirmatory testing of patients with suspected HPS who had visited Yosemite.

Commercial diagnostic laboratories that detected hantavirus antibody in submitted serum specimens were asked to notify and forward specimens to state public health laboratories or CDC for confirmatory testing.

State health departments that identified the death of a previously healthy person who had a history of travel to Yosemite were asked to collect tissue specimens for confirmatory testing by CDC.

CDPH and NPS issued media releases recommending that persons who experienced febrile illness after travel to Yosemite consult with their health care provider about possible hantavirus infection.

Medical records for laboratory-confirmed HPS patients were reviewed for relevant clinical information. Patients with confirmed cases or a proxy family member if the patient was deceased and their non-ill travel companions who had shared lodging with the patient were interviewed by telephone, using a standardized questionnaire.

One non-ill companion was interviewed for each patient identified. By using closed and open-ended questions, patients and non-ill companions were asked about demographic characteristics, illness history, travel, and other activities in the weeks preceding illness onset.

Participants were asked about the type of lodging and activities they experienced during their Yosemite visit, particularly actions e. Environmental Investigation Yosemite lodgings where patients had stayed and other nearby buildings were visually evaluated for evidence of active or recent rodent activity e.

Rodent populations in and around Yosemite guest lodgings were live-trapped for evaluation. Sherman live-traps H.

Rodent abundance was estimated by trap success, defined as the ratio of captured mice to the total number of traps set.

Captured rodents were anesthetized, euthanized, measured, and identified to species. Results Laboratory, Clinical, and Epidemiologic Findings Ten case-patients were identified among persons who had visited Yosemite overnight during summer The median age of the 10 patients was Salient case-patient clinical characteristics are displayed in the Table.

Eight patients had respiratory signs or findings compatible with HPS, 3 of whom died. The illnesses of 2 case-patients did not fulfill the complete HPS definition.

One patient had been examined in an emergency department for fever, cough, and shortness of breath and was discharged and recovered.

The second patient did not report fever or pulmonary symptoms but had severe headache, nausea, vomiting, and diarrhea.

Both patients were tested for SNV retrospectively after they heard about the Yosemite outbreak in the news and had detectable antibodies against SNV.

The onset of illness for the 10 case-patients occurred from July 2 through August 16, Figure 1. From interviews with case-patients or their proxy and non-ill companions , we found that case-patients reported engaging in activities and behavior similar to those of non-ill companions while at Yosemite, and no single activity typically associated with risk for HPS was common among patients.

Clinicalcharacteristicsof10hantaviruscase-patients whowereexposedatYosemiteNationalPark, Patientcharacteristic No.

One of these patients stayed in the same cabin in which another patient had lodged 13 days earlier.

In short, in some details near, the baby Mort is a child as others, if it is only he has a future any line: when he will be big.

Editions Delcourt When Tate Collins meets Miles Bowman, she knows that it is not the thunderbolt. And with good reason, the nice pilot has time only as short-lived adventures.

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Editions France Leisures When Be teeming takes its plane for New York, she is far from imagining that her trip is going to take a strange turn.

In the middle of flight, she is approached by a strange creature which asks her if it accepts the inheritance of an aunt disappeared for 20 years.

The trouble prowls always in these accursed mountains. Will they manage to avoid him? Raised on a summit arid and frozen, a man in high stature gets ready for the ceremony of the sacrifice.

Very far under him, the whole village keeps its breath by considering him. A good novel with a searched world and a nice writing.

I really wanted to follow the adventures of the ancestors of the heirs, even if I really did not expect in this by reading the summary.

I am going to follow adventure shortly. All those who carry Mark. Look at these young people. Here is your only family, currently.

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An Africain princess must confront the desert and the mountain of the goddess-snake to claim her inheritance.

To protect himself from passed trials, Rylee Thomas is always in the control of herself until the day when she meets the only man who, just, could give him desire to lose grip.

Colton Donavan, a superb, arrogant and dark boy, taught to get all what he. Alice and Oliver Ryan are the picture of conjugal happiness.

Accomplices, lover, they lead the life of ease. However, one evening, Oliver attacks Alice with such violence as he plunged him into the coma.

While everybody tries to understand the reasons of this act of a brutality without. For as long as the memories go back up, Andraste sees hidden from the eyes of the world.

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What is the price of a life? The day before the Normandy landings, facing certain death, what would you be ready to promise to exchange your place?

And what will be worth this promise, after the war, while all witnesses will have died or disappeared? When a young widow, Alice.

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And possibly my last, as this could be a co. I have thought for a long time that a novel for teenager was; little like the Old man and the sea of Hemingway, which they read or which they make read to the secondary school pupils to build them, to show them an example of courage and of abnegation - certainly objectively useless; but rest honour.

To tell the truth the leader of work of Herman Melville, published in , is a so considerable book as of a peculiar building, alternating romantic narration and technical parts, chapters and paragraphs constructed a bit like a textbook of whaling, or else, sometimes, of a biology treaty on Cetaceas; book of observations and of collections of scientific data which would not have undoubtedly disavowed Darwin.

But I will forget almost the style grabbing with Melville. Because a work is which leaves nobody uninterested; with a prose put down between the space of the sea by day without wind and the ugly storms where the black of soul leaves feed the madness of the men; a wild drunkenness where the marine monsters come to haunt drinking sessions on bottom of whale oil, of revenge and of redemption.

Some, I have just discovered him at instant, will go to search the marvellous of Moby Dick in one. Those there are the merchants mislaid by an ocean of points, these ship charterers who always stay in quay.

Let us pass. It is to tell the truth by a reading, there is some years it on the radio, that I discovered this literature monument.

Immediately the harpoon of fascination was planted in my skull. A certainly love-hate fascination, the changeable flame of which did not cease eating of its own blackness, its own bankruptcies.

Because Moby Dick is the history of irremediable one bankruptcy and which import if they abandon the idea of drawing the outline or of expressing grandiose sourness.

Words go off, hung on this rope linked up with the body of the monster which gets ready to dive into abimes. A fate about which they do not know if it is desirable to that of consenting martyrs of circus games in ancient Rome.

But these sailors, for the luckiest, will be able to find consolation in the idea - vainglory to tell the truth - which they at least will leave for vanishing trace of their passage on this earth of pain and of labour, a plate in the church of Nantucket.

Melville infused Moby-Dick with a power of expression he had not previously possessed. Moby-Dick, written in , recounts the adventures of the narrator Ishmael as he sails on the whaling ship, Pequod.

Herman Melville August 1, — September 28, Moby dick it e st also and especially his characters. But not in Melville's novel.

Undoubtedly that in one situation extremes curls up some size, height as well in courage as in abjectness.

Everything begins in the inn of the Prompter, on a night of snow and of wind in this located island off the Cape r will go to put down its brushes.

Having passed fear and one night without sleep, soon Queequeg will become his soul mate:. See the world! The other who does not make a mistake invites then there the young man to go up to the ship's rail and to glance on the side of the wind, and to ask him what he sees.

Cannot you see the world from there where you are. Also will make Queequeg, whose initialling consists merely of a small schematic fish, a kind of eight put to bed such as draw it the children.

The ship is Pequod. Later, aboard, at night once put to bed, they will hear the foot of sinister wood of Achab to beat the bridge.

And so of next day, and of day afterwards. But languages untie themselves and rumour begins painting the outline of the vengeful madness of the one-legged person.

On the boat there is also Indian and Negro as companions of the cannibal in harpoons. He will have Hot toddy measure is enough of a hellish toast!!

Who hit you only by the most blind of instincts! Ce will be vain. But I am not going to narrate all history here; she is only beginning.

I will say nothing of yellow, peculiar stowaways souquant to close to the whale, neither of grey and of pallid, nor of cryptic turbaned sicarian who is held silent in side of Achab.

Nothing of this kitchen of the Hell, and all this oil - so much oil, so precious. Without counting these pisseuses barrels, reasons of direct confrontation between Achab and its second.

This last wants that stopover is made to stop them. But the captain has only one idea at the head: Moby Dick.

And enduring no insubordination its gun goes out, which it checks off on the belly of Starbuck. How is it possible to stalk the single animal in such unlimited immensity?

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Infinite waiting and routine of these endless days on a sea stall will be so felt there; deaf anxiety also near action, flash finally of tracking.

The madness of the men, the force of the animal. He will have been understood, I liked this comic strip and waits for continuation not without impatience.

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Coccidioides immitis 8 Rhizopus arrhizus Soft tissue, sepsis Soft tissue Pulmonary;6 3. Thailand 10 Garzoni et al.

Thailand 11 Gunaratne Colombo, etal. Southeast 15 Asia Schneider et al. Colombia 17 Flynnetal. Disaster-associated fungal infections are similarly uncommon, but they are becoming increasingly recognized and are typically attributable to the impact phase of a disaster because such infections primarily result from inhalation or cutaneous inoculation of fungal spores directly from the environment 3,4.

During a disaster, pathogenic fungi can be displaced from their natural habitats, which could increase their environmental concentration or introduce them to areas where they would not normally be found, resulting in contact with injured persons and potentially causing fungal infections.

To increase awareness of these events among health care providers and public health officials, we summarize the known occurrences of fungal infections associated with natural disasters Table.

Reportsdescribing cases of post-disaster fungal colonization without infection are not included in the table. Pathogenic fungi exist in a broad range of natural habitats but are believed to be more common in subtropical and tropical areas of the world, probably because of environmental restrictions on their growth or propagation Known geographic habitats of some pathogenic fungi for example, Blastomyces, Coccidioides, and Histoplasma are well defined, but others such as Aspergillus and other molds are thought to be ubiquitous.

The abundance and distribution and therefore, potential to cause disease of environmental fungi probably depend on climatic or environmental factors such as ambient temperature and moisture Examples include coccidioidomycosis incidence in Arizona, which has been shown to correlate with hot, dry conditions; blastomycosis clusters observed in association with rainfall after periods of decreased precipitation; and incidence of Penicillum marneffei penicilliosis, an opportunistic infection which is endemic to Southeast Asia and increases in incidence during rainy months 21, Aspergillosis and other invasive mold infections have also been postulated to correlate with seasons or weather patterns in some geographic areas Although seasonal changes and weather effects probably play a role in the growth and distribution of many pathogenic fungi, environmental disruption is a key factor in the dispersal of these organisms and their resulting potential for causing infection.

Natural disasters can cause large-scale disruption of fungal habitats, which can lead to clusters of respiratory, cutaneous, or other forms of fungal disease.

Studies of clinical specimens collected from persons injured during disasters highlight the diversity of potential fungal pathogens in the natural environment.

For example, after an 8. Similarly, after a tornado in Texas, United States, fungi were identified in 8 6. In each of these reports, Airborne Inhalation is a common route for fungal infections.

Fungi are known to cause respiratory infections ranging from asymptomatic to life-threatening, depending on the pathogen and host characteristics.

Coccidioides spp. Two instances of disaster-associated coccidioidomycosis have been described. An outbreak of coccidioidomycosis after the January earthquake in Northridge, California, United States, was 1 of few known examples of any infectious disease outbreak directly related to a geophysical disaster 3.

Coccidioides spores were presumably aerosolized as a consequence of the earthquake, its aftershocks, and associated landslides and were dispersed by the resulting widespread dust clouds In Ventura County, California, outbreak-associated coccidioidomycosis cases were identified, and investigators found that dust exposure was substantially associated with acute illness Another coccidioidomycosis outbreak occurred after a severe dust storm in the southern San Joaquin Valley of California in December The storm originated near Bakersfield, an area to which coccidioidomycosis is highly endemic, and covered nearly 90, km2, an area larger than the state of Maine In Sacramento County, an area to which the disease was not previously considered to be endemic, cases of coccidioidomycosis were attributed to the dust storm, including 16 cases of disseminated disease Eighteen additional cases were identified at a US Navy air station in Kings County 19 , and other California counties affected by the storm saw more coccidioidomycosis cases than usual; for example, Kern County recorded cases during January and February , compared with 17 cases during those months in the previous year Near-drowning Drowning and near-drowning are common during and after disaster-related flooding 1.

Tsunami lung can be caused by bacteria, fungi, or both. Other fungal pathogens, such as Aspergillus, have also been implicated as agents of tsunami lung; after the Great East Japan Earthquake and subsequent tsunami, a previously healthy near-drowning victim who later died was found to have pneumonia caused by Aspergillus fumigatus and evidence of multiorgan disseminated aspergillosis upon autopsy 5.

The report describes delays in specimen transportation and receipt of culture results caused by the aftermath of the earthquake, which led to a delay in diagnosis and treatment 5.

In Sri Lanka, acute respiratory issues attributed to post-aspiration pneumonitis and polymicrobial pneumonia that were not related to communicable illnesses were the most frequent medical problems after this disaster In Banda Aceh, Indonesia, several patients with necrotizing pneumonia, who did not respond to broad-spectrum antibacterial drugs, probably had polymicrobial infections that may have included fungal organisms However, limited diagnostic capacity for fungi may have affected the ability to identify the potential role of fungi in these infections.

A report from Germany demonstrated that among a cohort of 17 tourists injured Figure. A left flank wound in a mucormycosis casepatient, with macroscopical fungal growth tissue with white, fluffy appearance and necrotic borders before repeated surgical debridement.

Copyright Massachusetts Medical Society. Reprinted with permission. Soft Tissue Fungal Infections after Disasters The risk for wound infections after a natural disaster is high when wounds are contaminated with water, soil, or debris In addition, damage to the local health care infrastructure can compromise the ability to properly irrigate contaminated wounds with sterile solution or promptly treat injured persons with topical or systemic antimicrobial drugs These factors can result in severe, often polymicrobial, infections of otherwise relatively minor injuries Although most documented disaster-associated soft tissue infections are bacterial typically gram-negative pathogens such as Aeromonas, Escherichia coli, and Klebsiella 33 , fungal wound infections can also occur, and they could be under-recognized because they can be clinically similar to bacterial infections, particularly during the early stages of infection.

Mucormycosis, caused by fungi that belong to the order Mucorales, is perhaps the most recognized example of post-disaster fungal soft tissue infection.

According to a report of 38 patients with necrotizing lesions who were hospitalized after the volcano, 8 patients had infections caused by the mucormycete Rhizopus arrhizus oryzae Similarly, a cluster of mucormycosis cases caused by Apophysomyces trapeziformis occurred among 13 persons who were severely injured in the May 22, , tornado in Joplin, Missouri, United States 4.

Penetrating trauma and an increased number of wounds were shown to be independent risk factors for mucormycosis. Whole-genome sequencing of A.

These reports illustrate some of the clinical challenges associated with soft tissue mucormycosis, caused by organisms that may initially appear indistinguishable from other types of wound infections but require aggressive treatment with intravenous antifungal medication and surgical debridement 13, Other agents of fungal soft tissue infections in survivors injured during the Indian Ocean tsunami include Fusarium which later caused systemic infection in 1 tourist 15 and Cladophialophora bantiana in 2 other tourists Subcutaneous C.

Soft tissue fungal infections have also been documented among persons who were not directly injured during a disaster but who sustained minor trauma while performing post-disaster tasks: in Texas after Hurricane Ike in , chromoblastomycosis was diagnosed in 3 patients, all of whom had histories of cancer and all of whom described clearing brush and fallen trees near their homes after the storm 8.

An outbreak of Aspergillus meningitis after the Indian Ocean tsunami was associated with the use of spinal anesthesia for cesarean section infant delivery for 6 previously healthy women in Sri Lanka The first 5 case-patients were initially treated for bacterial meningitis, but the discovery of Aspergillus during the post-mortem examination of the index casepatient led to the use of amphotericin B and voriconazole in the surviving case-patients Investigation of various medical supplies revealed that syringes from a central storage facility were contaminated with A.

Indoor Mold Exposures after Disasters Disaster-induced water damage to structures can create moist environments that can promote indoor fungal growth, but the extent to which damp indoor spaces and mold growth affect human health remains somewhat ambiguous A report by the Institute of Medicine found sufficient evidence of association between indoor mold exposure and upper respiratory tract symptoms, cough, and wheezing, and evidence of an association between indoor mold exposure and some noninfectious health conditions that included asthma symptoms in persons with asthma and hypersensitivity pneumonitis in some groups of people Although the report found no association between indoor mold exposure and invasive infection in healthy persons, there was evidence to support a link between exposure to Aspergillus and aspergillosis in severely immunocompromised persons Few data clearly demonstrate that indoor mold exposures increase the risk for invasive infection in post-disaster settings.

Despite these high levels of indoor mold growth documented in some areas, 1 study showed no elevated risk for fungal infections among immunocompromised patients exposed to water-damaged buildings after Hurricane Katrina; 1 patient, 1.

Colonization in the absence of related clinical symptoms was observed in persons who returned to their water-damaged homes after Hurricanes Rita and Katrina: the mucormycete Syncephalastrum was detected in various clinical specimens from 8 persons whose selfreported exposures to mold ranged from none to heavy, but none had evidence of invasive infection After the Great East Japan Earthquake and subsequent tsunami, a medical relief team observed unexplained chronic cough among a group of previously healthy persons living in a temporary refuge Fungal cultures of sputum samples from 6 persons yielded Aspergillus fumigatus, A.

Disasters, Fungi, and Global Climate Change Climate change could be affecting the ecology of pathogenic fungi in ways that are not yet fully understood; even minor or gradual changes in temperature, moisture, and wind patterns might affect fungal growth, distribution, and dispersal For example, warmer average global temperatures may allow the geographic range of fungi typically restricted to tropical and subtropical environments, such as Cryptococcus gattii, to expand into areas that are currently more temperate Global warming has also been hypothesized to select for fungi with tolerance to warmer temperatures Huppert and Sparks suggest that global climate change is contributing to greater frequency and severity of extreme weather events and that current patterns of population growth, urbanization, and human activity create conditions that render many communities increasingly vulnerable to these hazards Coupled with an increased risk for natural disasters, a larger or more geographically widespread ecologic burden of pathogenic fungi could lead to greater numbers of disaster-associated fungal infections through any of several mechanisms: inhalation of spores dispersed as a result of geophysical disruption, traumatic implantation of fungi into wounds contaminated with organic matter, or infection associated with suboptimal medical care where the local health care system has been damaged or destroyed.

Conclusions Disasters are complex events that can result in a wide range of health effects, although infectious disease outbreaks as an immediate consequence of disasters are uncommon.

These infections can occur in persons who do not have the typical immunocompromising risk factors for fungal infection but who have experienced near-drowning, trauma, or other unusual exposure to the environment, such as a dust storm.

A fungal infection should be considered early if a patient has a persistent or progressive infection that is not responding to initial antibacterial treatment, particularly because rapid diagnosis and administration of appropriate antifungal therapy can improve patient outcomes.

Prompt restoration of disaster-affected aspects of the local health care infrastructure may help facilitate earlier diagnosis and treatment and possibly reduce the risk for infection associated with the use of contaminated medical equipment or substandard care.

Strategies to reduce disaster-associated fungal infections should be considered within the broader context of comprehensive and sustainable risk reduction methods to prevent disasterrelated injury and illness.

Her interests include the epidemiology of fungal infections and health communications. His research interests include the prevention and epidemiology of fungal infections.

References 1. Noji EK. The public health consequences of disasters. Prehosp Disaster Med. World Health Organization.

WHO definitions: emergencies [cited Aug 12]. Negligible risk for epidemics after geophysical disasters. Emerg Infect Dis. Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in N Engl J Med.

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Subcutaneous phaeohyphomycosis caused by Cladophialophora bantiana. Arch Pathol Lab Med. Damp indoor spaces and health.

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J Occup Environ Med. Extreme natural hazards: population growth, globalization and environmental change. Warnock DW. Fungal diseases: an evolving public health challenge.

Alam, Thomas A. Weppelmann, Chad D. Weber, Judith A. Johnson, Mohammad H. Rashid, Catherine S. Birch, Babette A. Brumback, Valery E.

Madsen Beau de Rochars, J. Glenn Morris, Jr. Cases have continued to occur, raising the question of whether the microorganism has established environmental reservoirs in Haiti.

Toxigenic Vibrio cholerae O1 El Tor biotype strains were isolated from 3 1. All samples containing V.

Our data substantiate the presence of toxigenic V. These isolations may reflect establishment of long-term environmental reservoirs in Haiti, which may complicate eradication of cholera from this coastal country.

E pidemic cholera was identified during October in Haiti; initial cases were concentrated along the Artibonite River 1,2.

Because cholera had not been reported in Haiti for at least years, there is a high likelihood that the responsible toxigenic Vibrio cholerae strain was introduced into Haiti, possibly through Nepalese peacekeeping troops garrisoned Author affiliations: University of Florida College of Public Health and Health Professions, Gainesville, Florida, USA M.

Alam, T. Weppelmann, V. Madsen Beau de Rochars, A. Weppelmann, C. Weber, J. Johnson, M. Rashid, C. Birch, B. Brumback, V. Morris, Jr.

Rashid, V. In the months after October , cholera spread quickly through the rest of Haiti: , cases and 7, deaths were reported in the first year of the epidemic 1.

In the intervening years, cases and epidemics have been reported, and it has been suggested that onset of the rainy season serves as a trigger for disease occurrences 2,8.

In our studies in Peru 16 , water temperature was found to be the primary trigger for these environmental blooms and could be correlated with subsequent increases in environmental counts and occurrence of human illness.

To understand patterns of ongoing cholera transmission and seasonality of cholera in Haiti, and to assess the likelihood of future epidemics, it is essential to know whether environmental reservoirs of toxigenic V.

We report the results of an initial year of monitoring of environmental sites in the Ouest Department of Haiti, near the towns of Leogane and Gressier, where the University of Florida Gainesville, FL, USA has established a research laboratory and field area.

Sites were selected along transects of 3 rivers in the area and at 1 independent estuarine site: the Momance River 4 up-river sites and 1 estuarine site at the mouth of the river , the Gressier River 4 up-river sites and 1 estuarine site at the mouth of the river , the Tapion River 4 river sites , and an independent estuarine site at Four-a-chaux, which is a historic ruin and tourist attraction.

Up-river sites on the Momance and Gressier Rivers were in the Figure 1. Locations of environmental sampling sites near the towns of Gressier and Leogane in Haiti.

A Number of Vibrio cholerae O1 isolates obtained from sampling sites. The number of V. A total of samples were collected for culture for V.

Isolation and Identification of V. In addition to the conventional sample enrichment technique 18 , we used alkaline peptone water APW to enrich water samples.

Each colony was examined by using the oxidase test; oxidase-positive colonies were tested by using V.

The samples were collected from 14 environmental sites. One gram of the sample was mixed with mL of saline and then homogenized in a sterile blender; 1.

Genetic Characterization of V. Rainfall estimates were based on National Aeronautics and Space Administration data for the study region bounded by the rectangle Data Analysis We examined the effects of water quality factors on the presence of toxigenic and nontoxigenic V.

Stratification excluded sites that had all-positive or all-negative outcomes; of the remaining sites, regression analysis showed O1 V.

Results V. Physical parameters for the environmental water samples are summarized in Table 3. Temperatures tended to increase as rivers approached the sea.

As shown in Figure 2, mean water temperature from all sites showed evidence of seasonal variation. Measurement of rainfall was available for the region as a whole Figure 3.

However, site-specific rainfall data were not available; consequently, rainfall was not included in the regression models.

Isolation of V. In a conditional logistic regression analysis with water quality factors Table 4 , the only variable that emerged as statistically significant was water temperature odds ratio 2.

As shown in Figure 3, there was evidence that V. Of samples, the only V. Non-O1 V. As observed with O1 strains, isolations were more common at the mouths of the rivers and in estuarine areas Figure 1, panel B ; however, the non-O1 strain was found farther upriver than were O1 strains and was isolated from several sites in the mountains.

Non-O1 strains were isolated from all sites that were also positive for O1 strains. Non-O1 strains were isolated in all months, without an obvious association with regional Table1.

Discussion Before this study, isolation of 2 toxigenic V. In contrast, we isolated ctx-positive and ctx-negative V.

Our successful isolation of the microorganism from the environment may reflect localization of environmental isolates near Gressier and Leogane, where our study was conducted; however, we believe that our findings are more likely to be a reflection of the method used.

Data presented here suggest that, in addition to conventional APW enrichment, longer APW enrichment time and enrichment at higher temperatures contributed to an increased rate of isolation of V.

We also note some issues relating to sample transport: Baron et al. As has been reported, Vibrio spp. Water from which we isolated V.

Water temperature was found to be the single physical parameter that was substantially associated with isolation of these organisms; higher temperatures were concentrated downriver and in estuarine areas.

For our analysis, we used a conditional logistic regression model to permit stratification by site. Although we found very low numbers for V.

In our studies of aquatic animals likely to be eaten by humans, we did isolate V. The isolate was nontoxigenic; consequently, its association with disease is unclear.

After analyzing the results of this study, we asked the following question: has V. Toxigenic V. Although data are limited, there was at least a suggestion that isolation of V.

We also found non-O1 strains widely distributed throughout the environment, including mountain river sites, consistent with widespread dissemination in environmental reservoirs.

Although we cannot be certain that O1 and non-O1 strains grow under comparable conditions, the clear establishment of non-O1 V.

We propose that the 3 isolates that are positive for ctx genes be classified as circulating V. To better understand the evolutionary mechanisms involved, we are performing further sequence analysis of clinical and environmental strains.

Conclusions The apparent introduction of toxigenic V. If these O1 strains establish stable environmental reservoirs in Haiti, in the setting of ongoing problems with water and sanitation, there is a high likelihood that we will see recurrent epidemics Table4.

These circumstances clearly have implications for current plans by the Haitian Ministry of Public Health to eradicate cholera in Haiti within a decade The proposed implementation of vaccination programs and efforts to improve water supplies and sanitation will undoubtedly reduce case numbers, but as long as the causative microorganism is present in the environment, eradication of the disease will not be possible.

Establishment of environmental reservoirs and recurrent epidemics may also serve as a potential source for transmission of the disease to the Dominican Republic and other parts of the Caribbean 1.

Ongoing monitoring of potential environmental reservoirs in the areas near Gressier and Leogane as well as in sentinel sites throughout the country will be necessary to assess this risk and to permit development of rational public health interventions for cholera control.

Acknowledgments We thank Mohammad Jubair for his technical help with this study. His research interests focus on the ecology and epidemiology of V.

Spatio-temporal dynamics of cholera during the first year of the epidemic in Haiti. Recent clonal origin of cholera in Haiti. The origin of the Haitian cholera outbreak strain.

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti.

Enserink M. Cholera linked to U. High-frequency rugose exopolysaccharide production by Vibrio cholerae. Appl Environ Microbiol.

Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation.

Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation.

Colwell RR, Huq A. Vibrios in the environment: viable but nonculturable Vibrio cholerae. Vibrio cholerae and cholera: molecular to global perspectives.

Morris JG Jr. Cholera in Lima, Peru, correlates with prior isolation of Vibrio cholerae from the environment.

Am J Epidemiol. Critical factors influencing the occurrence of Vibrio cholerae in the environment of Bangladesh. An evaluation of alkaline peptone water for enrichment of Vibrio cholerae in feces.

Southeast J. Public Health. Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor.

Microbiol Immunol. Molecular diversity of CTX prophage in Vibrio cholerae. Lett Appl Microbiol. National Aeronautics and Space Administration.

NASA earth data. Haitian Ministry of Public Health and Population. Daily reports of cholera cases by commune. May [in French] [cited May 14].

Lysogenic conversion by a filamentous phage encoding cholera toxin. Toxigenic Vibrio cholerae O1 in water and seafood, Haiti.

No evidence of significant levels of toxigenic V. PLoS Curr. Influence of water temperature, salinity, and pH on survival and growth of toxigenic Vibrio cholerae serovar O1 associated with live copepods in laboratory microcosms.

Clinical and environmental isolates of Vibrio cholerae serogroup O carry the CTX phage and the genes encoding the toxin-coregulated pili.

J Clin Microbiol. Davidson, Sonia Agrawal, Moira L. Aitken, Shamira Shallom, Nabeeh A. Daugherty, Claire M. Fraser, Barbara A. Brown-Elliott, Richard J.

Wallace Jr. Holland, Elizabeth P. Sampaio, Kenneth N. Olivier, Mary Jackson, and Adrian M. Zelazny Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp.

Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil.

We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates.

Our findings highlight the necessity of identifying M. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.

Tettelin, S. Agrawal, E. Hine, S. Parankush, Q. Su, S. Daugherty, C. Davidson, N. Hasan, M. Shallom, S. Holland, E.

Sampaio, K. Olivier, A. Calado Nogueira de Moura, M. De Groote, M. Brown-Elliott, R. Previous studies have indicated great diversity within M.

However, suspicion of patient-to-patient transmission arose with the recent report of an outbreak of respiratory infection with M. The index case-patient and 4 additional patients all had multidrug-resistant isolates with resistance to amikacin and clarithromycin.

In a separate, recent study, whole-genome sequencing and epidemiologic analysis provided strong support for patient-to-patient transmission in 2 clustered outbreaks of M.

Isolates from both clusters showed resistance to clarithromycin, and isolates from one of the clusters also had mutations conferring resistance to amikacin.

The availability of whole-genome sequences from different M. We found high-level relatedness among strains from the 2 geographically distant outbreaks in Seattle and Papworth.

We also identified shared and unique genomic traits for strains from both cystic fibrosis outbreaks and for those from an outbreak of soft tissue infections in Brazil.

Materials and Methods Sequence Analysis of Outbreak Strains A subset of 6 isolates 2u, 12c, 14h, 19f, 20h, and 28c representing the breadth of genomic diversity observed within the Papworth cystic fibrosis outbreak clusters 1 and 2 6 were selected.

Illumina sequencing reads from each of these isolates were assembled into sets of contigs by using Velvet software These contigs were combined with draft genome sequences of the Seattle cystic fibrosis outbreak and available whole-genome sequences of M.

Core segments of the alignment that are shared among all isolates included in the analysis were identified and concatenated by using Phylomark software Concatenated nucleotide sequences, including singlenucleotide polymorphisms SNPs , were then used for construction of a neighbor-joining phylogenetic tree by using MEGA software The use of microbial samples and data was approved by the ethics committees at each of the institutions involved.

To replicate data from the Papworth cystic fibrosis outbreak clusters 1 and 2 6 by using a similar approach, we mapped sequencing reads from the subset of 6 Papworth isolates, together with reads with from the 3 Seattle cystic fibrosis isolates and soft tissue strain CRM from Brazil Table 1 , onto the M.

The resulting tree replicated the topology of clusters 1 and 2 and showed that the Seattle isolates are most closely related to cluster 2.

Published forward primers for cya and gdhA 30 did not amplify in silico for some M. Alleles from each gene were Table 1. Results Phylogenetic Characteristics of Outbreak Strains A core genome phylogenetic tree Figure 1 showed a tight cluster of the 3 Seattle cystic fibrosis outbreak strains.

The Seattle cystic fibrosis cluster was closely related to the 2 cystic fibrosis clusters described for the Papworth outbreak 6 and the Birmingham, UK, cystic fibrosis isolate 47J26 9.

Furthermore, the Seattle and Papworth cystic fibrosis outbreak strains showed some relatedness to strains CRM and GO derived strains known collectively as BRA isolated during an epidemic of soft tissue infections in Brazil 32 and the M.

The cumulative size of core segments of Mugsy alignments provides information on relatedness among groups of strains compared.

The core genome reduces in size as more genomes are added; an expected major decrease occurs after addition of more distant strains to the group.

The average genome size of cystic fibrosis outbreak strains was 4. As expected, including unrelated available clinical M.

Further addition of M. Strain GO 06 was excluded from the analysis because its genome harbors a large number of ambiguous nucleotides and an unusual hybrid appearance with fragments of M.

Strains 47J26 and M18, isolated from the sputum of a cystic fibrosis patient in Birmingham, UK, and a lymph node sample from a patient in Malaysia, respectively, were related to the outbreak strains Figure 1.

Neighbor-joining phylogenetic tree based on whole-genome multiple alignment of 24 Mycobacterium abscessus group genomes.

Genomes in Table 1 were aligned by using Mugsy 22 , core segments of the alignment were identified by using Phylomark 23 , and resulting concatenated nucleotide sequences were used for construction of the midpoint-rooted neighbor-joining phylogenetic tree by using MEGA Strains from an outbreak of M.

SNPs, single-nucleotide polymorphisms. However, no information was available about any epidemiologic link between cystic fibrosis strain 47J26 to reported or unpublished outbreaks, and no clinical information was available about the patient from whom strain M18 was isolated.

Therefore, both strains were excluded from the SNP analysis. Nevertheless, SNPs for these 3 strains at positions relevant to the outbreak strains are shown in the Technical Appendix wwwnc.

A total of identical SNPs in the core segments of Mugsy alignments were shared by the 10 outbreak strains but were different in available M.

Of the SNPs, 95 gave rise to nonsynonymous mutations in several genes, including virulence factors mammalian cell entry and yrbE proteins , transcriptional regulators TetR family , and lipid metabolism genes online Technical Appendix.

Sixteen SNPs were shared only by the 3 Seattle cystic fibrosis outbreak strains, including nonsynonymous mutations in a mycobacterial large membrane protein MmpL family involved in lipid transport and virulence 34 and genes involved in amino acid and energy metabolism Figure 2; online Technical Appendix.

Eighty-six SNPs were present only in strain CRM soft tissue outbreak from Brazil, including nonsynonymous mutations in an MmpL family protein; transcriptional regulators; and lipid, amino acid, and energy metabolism genes Figure 2; online Technical Appendix.

Alignment of the MmpL family protein with distinct MmpL proteins described above for the Seattle cystic fibrosis outbreak and the Brazil soft tissue outbreak showed diversity at several amino acid residues in all 3 proteins.

We also searched for polymorphisms associated with macrolide and aminoglycoside resistance. The Papworth Figure 2. Venn diagram of core single-nucleotide polymorphisms SNPs shared by outbreak localities.

Core segments of the Mugsy 22 alignment of the 20 Mycobacterium abscessus subsp. Strains 19f, 14h, 12c, and 28a, representative of Papworth cluster 1, and Seattle strains shared the AG mutations in 16S rRNA, which conferred aminoglycoside resistance In the first approach, we retrieved rpoB sequences from the 6 genomes of representative strains of the Papworth cystic fibrosis outbreak and performed partial sequencing of the rpoB gene for selected isolates from the Seattle cystic fibrosis outbreak.

We then compared these sequences with those of isolates from the outbreak in Brazil and unrelated clinical isolates comprising M. However, none of the M.

Most of the 26 M. However, 4 strains harbored this signature Table 2 29, Multiple alignment of rpoB sequences among available M.

Multiple alignment of secA1 sequences among available M. Further analysis of secA1 sequences from 12 M. Those 2 strains were included among the 4 strains that had the 2-SNP rpoB signature.

We also developed a simple MLST protocol that could be used as a second confirmatory assay. Alleles for each of 13 housekeeping genes cya, gdhA, argH, glpK, gnd, murC, pgm, pknA, pta, pur, rpoB, hsp65, and secA1 were extracted and concatenated for each M.

The Seattle and Papworth cystic fibrosis outbreak strains grouped together in the tree with cystic fibrosis strain 47J26 and isolate M18 from Malaysia Figure 3.

Thus, partial sequencing of rpoB and secA1 gens, followed by target MLST analysis, could be used to rule out isolates as belonging to these 2 cystic fibrosis clusters.

Neighbor-joining phylogenetic tree based on target multilocus sequences types from 20 Mycobacterium abscessus subsp. Electronic PCR was performed on the M.

Nucleotide sequences from each gene were concatenated for each genome and aligned by using ClustalW 31 , and the core alignment was used for construction of a midpoint-rooted neighbor-joining phylogenetic tree by using MEGA The longer branch length for Papworth isolate 12c was caused by low-quality nucleotides single-nucleotide polymorphisms [SNPs] located at the edge of Velvet contigs.

Discussion The implications of this study are extensive. Currently, most experts recommend identifying isolates of M.

This report further corroborates these recommendations and places even greater pressure on clinical laboratories to fully identify M.

Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness 4,, nt core genome alignment size, 11 shared unique SNPs with each other and major-level relatedness 4,, nt core genome alignment size with soft tissue epidemic strains from Brazil.

Genomic features shared between strains from all 3 outbreaks might make them more transmissible, whether from patient to patient directly or indirectly as in cystic fibrosis outbreaks or from a common source, as in soft tissue infections.

We speculate that some of these specific genomic traits may be favorable for the successful establishment of epidemic soft tissue infections.

A previous study did not detect a common source or person-to-person transmission of the M. Our findings emphasize the necessity of screening all isolates of M.

Because of evidence supporting patient-to-patient transmission of multiple different respiratory tract organisms, the Infection Control Guidelines currently in draft form for public comment of the United States Cystic Fibrosis Foundation CFF www.

Patients with cystic fibrosis are advised not to attend indoor meetings with other cystic fibrosis patients CFF and Infection Prevention and Control Guidelines It remains unclear why intercontinental organisms are so closely related.

One hypothesis is that direct patient contact led to transmission. The Seattle index case-patient traveled to British Columbia, Canada, before and after acquiring mycobacterial infection, to Oregon before mycobacterial infection, and to Atlanta, Georgia, and Bethesda, Maryland, after mycobacterial infection.

However, the patient did not report any contact with other cystic fibrosis patients at these destinations.

A second hypothesis is that the mycobacterial strain could have been carried by persons with cystic fibrosis who were clinically well.

A third hypothesis is that there was an independent selection of M. Availability of additional whole-genome sequencing data tracking the global epidemiology of the M.

In addition, this data will help delineate global clusters of M. Addendum Recent whole-genome data show deep genetic separation of 3 subspecies, ruling against grouping M.

Acknowledgments We thank Josephine Bryant, Dorothy Grogono, Julian Parkhill, and Andres Floto for their help and for providing sample identification and accession numbers for the Papworth outbreak isolates.

H, and M. Carter Foundation. His primary research interests are the use of comparative and functional genomics to understand bacterial diversity and virulence, study host-pathogen interactions, and identify vaccine candidates and drug targets to cure disease.

Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel. Nontuberculous mycobacteria.

I: multicenter prevalence study in cystic fibrosis. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France.

Mycobacterium abscessus and children with cystic fibrosis. Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center.

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.

Genome sequence of an epidemic isolate of Mycobacterium abscessus subsp. Genome Announc. Complete genome sequence of Mycobacterium massiliense.

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Annotated genome sequence of Mycobacterium massiliense strain M, belonging to the recently created taxon Mycobacterium abscessus subsp.

Complete genome sequence of Mycobacterium massiliense clinical strain Asan , belonging to the type II genotype. Genomic insights into the emerging human pathogen Mycobacterium massiliense.

Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus.

Mol Microbiol. Sep 3 [Epub ahead of print]. Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.

Draft genome sequence of Mycobacterium abscessus subsp. Draft genome sequence of Mycobacterium bolletii strain M24, a rapidly growing mycobacterium of contentious taxonomic status.

Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs.

Genome Res. Mugsy: fast multiple alignment of closely related whole genomes. Phylomark, a tool to identify conserved phylogenetic markers from whole-genome alignments.

MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.

Mol Biol Evol. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.

Inaccuracy of single-target sequencing for discriminating species of the Mycobacterium abscessus group. Cohort study of molecular identification and typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii.

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus sensu lato strains. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

Nucleic Acids Res. Epidemic of postsurgical infections caused by Mycobacterium massiliense. Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil.

Future Microbiol. MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.

Chem Biol. The detection and sequencing of a broadhost-range conjugative IncP-1beta plasmid in an epidemic strain of Mycobacterium abscessus subsp.

Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus. Antimicrob Agents Chemother.

A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae.

J Infect Dis. New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identification of Mycobacterium massiliense with inducible clarithromycin resistance.

Clinical significance of differentiation of Mycobacterium massili ense from Mycobacterium abscessus. Lack of transmission of Mycobacterium abscessus among patients with cystic fibrosis attending a single clinic.

Heine, Glenn A. Marsh, Christopher C. Broder, and Lin-Fa Wang In recent years, the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health.

For example, Hendra virus HeV , a zoonotic paramyxovirus, was discovered in , and since then, infections have occurred in 7 humans, each of whom had a strong epidemiologic link to similarly affected horses.

As a consequence of these outbreaks, eradication of bat populations was discussed, despite their crucial environmental roles in pollination and reduction of the insect population.

We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse, human, and environmental health.

The HeV vaccine for horses is a key example of a One Health approach to the control of human disease. H endra virus HeV is an emerging zoonotic paramyxovirus for which natural reservoirs are the 4 species of flying fox Pteropus bats found on mainland Australia 1.

Middleton, J. Pallister, R. Klein, J. Haining, R. Arkinstall, L. Frazer, J. Bingham, D. Johnson, J.

White, A. Foord, H. Heine, G. Marsh, L. Feng, C. Huang, N. Edwards, M. Wareing, M. Elhay, Z. Each casepatient had a strong epidemiologic connection to similarly affected horses through exposure to equine secretions late in the incubation period, during terminal illness, or at the time of postmortem examination of infected animals 2 : no human case of HeV infection has been attributable to direct spillover from bats 3.

There is no licensed anti-HeV therapeutic drug for use in any species. However, gene copy numbers increased exponentially with the onset of fever, when viral genome could also be recovered from blood, oral secretions, urine, and feces 6.

Rapid progression of clinical signs, as observed in equine field cases of this disease, led to euthanasia of experimental animals on humane grounds.

Viral RNA was recovered from all tissues sampled at postmortem examination, and virus was reisolated from lung, brain, lymphoid tissues, and kidney 6.

In accordance with epidemiologic observations 2 , it was concluded that HeV-infected horses in the immediate presymptomatic or symptomatic stages of disease pose a high risk for transmission of HeV to humans.

This risk is then exacerbated because it is symptomatic horses that come to the attention of veterinarians, leading to various 1 These authors contributed equally to this article.

One of the sides of each pen was able to be moved in toward the horse on a ratchet mechanism, allowing staff close access to the horses, as required, over the side of the pen without the need for them to enter the pen itself Horses were fed a mixture of lucerne alfalfa and grass hay, concentrates, and specified fruit and vegetables.

On the day before HeV exposure, an indwelling jugular catheter was sutured in position, and an intrauterine temperature data-logger was placed into each horse.

The humane end point was defined as fever for up to 48 h accompanied by increased respiratory rate, dyspnea, depression, ataxia, or pressing the head against the side of the stall.

Euthanasia was conducted by intravenous injection of a barbiturate following sedation with intravenous detomidine and butorphanol.

Ferrets and guinea pigs used as controls in efficacy studies to confirm pathogenicity of the inoculum were housed in pairs in the BSL-4 facility, given species-appropriate dry rations and dietary treats, and provided with water ad libitum.

While in the BSL-4 animal room, staff wore fully encapsulated suits with an external air supply. These events were accompanied by a marked rise in the number of HeV-related media reports.

The reports had an increasingly politicized focus on the role and control of flying foxes as carriers of HeV 8 and a deemphasis of the critical role played by horses in HeV transmission to humans.

Heightened public awareness of the risk that infected horses posed to humans persisted and was paralleled by increased numbers of veterinarians leaving equine practice because of personal safety and liability concerns 9.

The considerable investment in education and improved infection control measures that had been implemented did not effectively mitigate perceptions around the risks associated with the routine veterinary care of horses The actual mechanism of HeV transmission from bats to horses is probably complex and dependent upon socioeconomic, environmental, and ecologic factors 11 , and there is currently no straightforward solution for preventing transmission.

Eradication of flying foxes would pose extraordinary operational challenges, notwithstanding attendant moral, ethical, and environmental issues, and eliminating the interface between bats and horses is impractical for periurban and rural communities.

The most direct approach for reducing the risk posed to humans by HeV-infected horses would be implementation of a strategy that will lead to suppression of virus replication in horses.

We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse and human health.

The emergence of several highly pathogenic zoonotic diseases in humans in recent years has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health.

All subsequent vaccines were formulated with clarified CHO cell culture supernatant that was then gamma irradiated.

The change of the expression system from F cells to CHO cells was driven by the need for higher antigen yields, and equivalence was supported by laboratory analysis of the expressed antigens from the 2 systems and a comparison study in ferrets.

Vaccine formulations used in efficacy studies are summarized in Table 1. Immunization All immunizations comprised two 1-mL doses administered intramuscularly 3 weeks apart, unless stated otherwise.

Overall, 4 efficacy tests were completed; 2 vaccinated horses were used in the first test, 3 were used in the second, 2 were used in the third, and 3 were used in the fourth.

For the 4 tests, a pathogenicity control for the inoculum was provided by 1 horse test 1 , 4 guinea pigs test 2 , 2 ferrets test 3 , and 2 ferrets test 4.

Exposure conditions for 3 additional unvaccinated control horses were equivalent to those used in both vaccinated horses and the inoculum-control horse and have been described 6.

Sample Collection and Analysis During efficacy studies, nasal, oral, and rectal swab samples; urine and feces samples; and blood samples in EDTA were collected from the horses before virus exposure and then daily until the animals were euthanized.

At postmortem examination, the following tissues were collected for viral genome detection, virus isolation, histopathology, and immunohistochemistry according to 15 : adrenal gland, bladder, brain including olfactory pole , cerebrospinal fluid, guttural pouch, heart, kidney, large intestine, liver, lung, lymph nodes bronchial, inguinal, intermandibular, mandibular, renal , meninges, nasal turbinates, ovaries, pharynx, small intestine, spinal cord, spleen, sympathetic nerve, trigeminal ganglion, and uterus.

The following analyses were conducted as described 15 : quantitative reverse transcription PCR for the detection of the HeV N gene, histology, immunohistology, serum neutralization test, and virus isolation.

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