Salmonella

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Salmonella
Praveen Rao, Sophia W. Riccardi, Danielle
Birrer Seminar in Nucleic Acids-Spring 2004 Prof.
Zubay
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Salmonella

• Overview
• History and Epidemiology
• Molecular Biology
• Clinical
• Weaponization

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Overview

• Salmonella is a rod-shaped, gram-negative,
  facultative anaerobe in the family
  Enterobacteriaceae

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Salmonella Taxonomy

• The genus Salmonella is divided into two species,
  S. enterica and S. bongori (CDC).
• Over 2000 strains are grouped into S. enterica.
  This species is further divided into six
  subgroups based on host range specificity, which
  also involves immunoreactivity of three surface
  antigens, O, H and Vi.
• All strains that are pathogenic to humans are in
  species S. enterica, subgroup 1 (also called
  enterica).
• For example, the correct taxonomic name for
  the organism that causes typhoid fever is
  Salmonella enterica ssp. enterica, serovar typhi.
  The simplified version Salmonella typhi.
• Taxonomy has been revised several times, due to
  the degree of DNA similarity between genomes.
• For example, In the U.S., another legitimate
  species name for enterica is choleraesuis.
  

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Other Facts

• Bacterium of 2501 identified strains, as of 2001.
  Many different diseases are caused by more than
  1,400 serotypes of this bacteria genus.
• Salmonella derived from Dr. Salmon, a U.S.
  veterinary surgeon, who discovered and isolated
  the strain enterica or choleraesuis from the
  intestine of a pig in 1885.
• They are ingested orally by contaminated food or
  water. Refrigeration prevents growth but does not
  kill bacteria. Heating at 57-60C or 134-140F
  has shown to be effective in killing the
  bacteria.
• Optimal growth 37C or 98.6F

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Disease-associated facts

• Salmonellosis Any of several bacterial
  infections caused by species of Salmonella,
  ranging from mild to serious infections.
• Two main kinds in humans enteric fever
  (typhoid and paratyphoid) and gastroenteritis
  (non-typhoidal).
• The main feature for S. diseases is the Type III
  Secretion System, a needle-like multi-protein
  complex that is associated with transferring
  toxic proteins to host cells.

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Principal habitats in different types of
Salmonella

• Their principal habitat is the intestinal tracts
  and bloodstream of humans, and in the intestinal
  tracts of a wide variety of animals.
• The WHO groups Salmonella into 3 types
• - Typhoidal (enteric) Salmonella
• (example S. typhi)
• ?causes typhoid and paratyphoid fever
• ?restricted to growth in human hosts
• ?principal habitat is in intestinal
  tracts and the bloodstream

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• - Nontyphoidal Salmonella (example S.
  enteritidis, S. typhimurium)
• ?prevalent in gastrointestinal tracts of a
  broad range of animals, including mammals,
  reptiles, birds and insects.
• ?cause a whole range of diseases in animals and
  humans, mainly gastroenteritis.
• ?usually transferred animal-to-person, through
  certain food products fresh meat, poultry, eggs
  and milk
• - fruits, vegetables, seafood
• ?house and exotic pets, contamination through
  contact with their feces

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- Salmonella mostly restricted to certain
animals, such as cattle and pigs
infrequently in humans if these strains
do cause disease in humans, it is often
invasive and life-threatening.
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Salmonella

• Overview
• History and Epidemiology
• Molecular Biology
• Clinical
• Weaponization

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History of Salmonella

• Some historical figures are believed to have
  been killed by
• Salmonella
• Alexander the Great died mysteriously in 323
  B.C. In 2001, a group of doctors at the
  University of Maryland suggested that S. was the
  cause of death, based on a description of
  Alexanders symptoms written by the Greek author
  Arrian of Nicomedia.
• Prince Albert, the consort of Queen Victoria,
  died of a Salmonella infection in 1861. During
  the Victorian era, an estimated 50,000 cases per
  year occurred in England.

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History

• Scholars working on the history of Jamestown,
  Virginia, believe that a typhoid outbreak was
  responsible for deaths of over 6000 settlers
  between 1607 and 1624.
• Typhoid Epidemic in the Spanish-American War
  (1898)
• - In all, 20,738 recruits contracted the
  disease (82 of all sick soldiers), 1,590 died
  (yielding a mortality rate of 7.7)
• - It accounted for 87 of the total deaths
  from disease.
• - A significant number of these deaths
  actually occurred at training areas in the
  southeastern United States.

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History

• Typhoid outbreak in British camps during the
  South African War (1899-1902)
• - more soldiers suffered from typhoid fever
  than from battle wounds.
• - British troops lost 13,000 men to
  typhoid, as compared to 8,000 battle deaths.
• - outbreak was largely due to unsanitary towns
  and farms throughout Africa, and polluted soil
  was washed into the network of streams and rivers
  during the rainy season.
• Epidemic potential during a war prominent because
  of the disposal problems of mens discharges.

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History

• Similar problems of sanitation occurred in urban
  areas. Many historic documents report about
  typhoid outbreaks in England
• - Most outbreaks that were reported could be
  traced back to unsanitary water supplies or
  polluted milk supplies.
• - Dr. William Budd (1811-1880) documented his
  observations, published them in the Lancet It
  was known then that polluted water can spread the
  disease. Budd urged for more disinfection and
  water treatment
• - reports show that in the nineteenth century,
  population seemed powerless against this disease
  even though they knew it was perfectly
  preventable.
• - with the introduction of piped and filtered
  water supplies in most urban areas, its
  prominence as a cause of death had diminished.

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Salmonella vaccine

• First preventive measure against Salmonella was
  discovered in 1896, as an antityphoid vaccine was
  developed by the British surgeon Almroth Wright.
• Vaccine consisted of heat-denatured, rudimentary
  killed whole-cell bacteria said to be highly
  effective.
• Early wars -Immunization known, but new
• -the minimum dosage had not been clearly
  refined
• British War Office
  authorized it on a voluntary basis only
  
• most soldiers refused to be
  immunized because of
• violent reaction
  following injection possible contraction
• Urban outbreaks opposition to any type of
  vaccination a way around the problem of
  sanitation and cleanliness. It was seen as a
  disease of defective civilization due to
  defective sanitation.

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Salmonella vaccine

• Between 1904-1914, the vaccine had become
  respectable, in the scientific as well as
  military world.
• Vaccine was successfully used during World War I
  to reduce the number of soldiers who died of
  enteric fever (S. typhi).

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• Bottling typhoid vaccine, 1944
• Division of Biologic Products, U.S. Army of
  Medical Department Professional Service Schools

• First typhoid inoculation, 1909 United States
  Army Medical School

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History in the U.S.

• Typhoid Mary Mallon was the first famous
  carrier of typhoid fever in the U.S.
• Some individuals have natural immunity to
  Salmonella. Known as chronic carriers, they
  contract only mild or asymptomatic disease, but
  still carry the bacteria in their body for a long
  time. These cases serve as natural reservoir for
  the disease.
• Approximately 3 of persons infected with S.
  typhi and 0.1 of those infected with
  non-typhoidal salmonellae become chronic carriers
  which may last for a few weeks to years.
• One such case was Mary Mallon, who was hired as a
  cook at several private homes in the new York
  area in the early 1900s.

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History Mary Mallon

• Mary Mallon caused several typhoid outbreaks,
  moving from household to household, always
  disappearing before an epidemic could be traced
  back to the particular household Mary was working
  in. All together, she had worked for 7 families,
  with 22 cases of typhoid and one death.
• She was finally overtaken by the authorities in
  1907 and committed to an isolation center on
  North Brother Island, NY. There she stayed until
  she was released in 1910, on the condition that
  she never accept employment involving food
  handling.
• But She was found to work as a cook and to cause
  typhoid outbreaks again. She was admitted back
  to North Brother Island, where she lived until
  her death in 1938.

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Recent outbreaks

• More recently reported outbreaks in the U.S.
  involve different kinds of Salmonella strains,
  predominantly S. enteritidis and S. typhimurium.
• In 1985, a salmonellosis (S. typhimurium)
  outbreak involving 16,000 confirmed cases in 6
  states by low fat milk and whole milk from one
  Chicago dairy.
• Largest outbreak of food-borne salmonellosis
  in the U.S. Investigations discovered that raw
  and pasteurized milk had been accidentally mixed.
  

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Oregon Intentional Contaminationof Restaurant
Salad Bars
In September of 1984, 10 area restaurants in The
Dalles, Oregon, were involved with outbreaks of
S. typhimurium
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Outbreaks

• January 2000 infant aged 1 month visited a
  clinic with fever and diarrhea. A stool specimen
  yielded Salmonella serotype Tennessee. One week
  before illness onset, the infant's family moved
  into a household that contained a bearded dragon
  (i.e., Pogona vitticeps).
• During June 2002, a child aged 21 months was
  admitted to a hospital with fever, abdominal
  cramps, and bloody diarrhea. Blood and stool
  cultures yielded Salmonella serotype Poona (from
  pet Iguana).

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Foodborne diseases

• WHO in 2000 that globally about 2.1 million
  people died of foodborne illness
• in industrialized countries, about 30 of people
  suffer from foodborne diseases each year around
  76 million cases occur each year, of which
  325,000 result in hospitalization and 5,000 in
  death.
• (WHO, 2002)

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Why do foodborne diseases emerge ?

• Globalization of food supply for example,
  multistate outbreaks of S. Poona infections
  associated with eating Cantaloupe from Mexico
  (2000-2002)
• Unavoidable introduction of pathogens into new
  geographic areas for example, vibrio cholerae
  introduced into waters off the coast of southern
  U.S. by cargo ship (1991).
• Travelers, refugees and immigrants exposed to
  unfamiliar foodborne hazards.
• Changes in microorganisms evolution of new
  pathogens, development of antibiotic resistance,
  changes in the ability to survive in adverse
  environmental conditions.

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Why do foodborne diseases emerge ?

• Changes in human population population of highly
  susceptible people is expanding more likely to
  succumb to bacterial infections.
• Changes in lifestyle Great amount of people eat
  prepared meals. In many countries, the boom in
  food service establishments is not matched by
  effective food safety education and control.

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Relative Frequency of the disease in the U.S.

• Estimate 2 to 4 million cases of salmonellosis
  occur in the U.S. annually (reported and
  unreported). Salmonella accounts for the
  majority of food poisoning cases in the U.S
• Latest numbers
• In 2002, a total of 32,308 cases were
  reported from health laboratories in 50 states.
• The national rate of reported isolates was
  11.5 per 100,000 population. Shows decrease of
  7 compared to 1992, slight increase of 2 from
  2001.

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Epidemiology

• The most commonly reported serotypes, in history
  and present
• - S. typhi
• - S. enteritides and S. typhimurium
• The top 20 serotypes accounted for 80 of all
  isolates reported in the U.S. in 2001.

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Top 15 Salmonella Serotype list in the U.S., 2001
Country, Institution, Biological origin Total Serot ped Rank Serotype Count of Total Serotyped
U.S.A., Centers of Disease Control, Control and Prevention-FDDB Epi, 2001, Human 31,675 1 Typhimurium 6,999 22.1
    2 Enteridites 5,614 17.7
    3 Newport 3,158 10
    4 Heidelberg 1,884 5.9
    5 Javiana 1,067 3.4
    6 Montevideo 626 2
    7 Oranienburg 595 1.9
    8 Muenchen 583 1.8
    9 Thompson 514 1.6
    10 Saintpaul 469 1.5
    11 Paratyphi B tartrate positive 466 1.5
    12 Infantis 440 1.4
    13 Braenderup 388 1.2
    14 Agona 370 1.2
    15 Typhi 343 1.1
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EpidemiologyS. typhi (typhoidal Samonella)

• Causes enteric fever
• Have no known hosts other than humans.
• Transmission through close contact with infected
  or chronic carriers. While direct
  person-to-person transmission through the
  fecal-oral route is rare, most cases of disease
  result from digestion of contaminated food or
  water.
• Since improvements in food handling, piped and
  filtered water supplies as well as water/sewage
  treatment have been made, enteric fever has
  become relatively rare in developed countries.

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• However, typhoid fever is still a big
  health-problem in developing countries.
• The WHO estimates that there are worldwide about
  16 million of clinical cases annually, of which
  about 600,000 result in death. In comparison,
  about 400 cases occur each year in the U.S., and
  70 of these cases are acquired while traveling
  internationally.

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Salmonella typhi in developing countries

• Contaminated water is a common cause in the
  spread of typhoid fever. At the time of rain,
  the contaminated surface water further
  contaminates water supplies.
• Severity, Morbidity and complication rate is much
  higher than in Europe and North America due to
  lack of antibiotics supply, water filtration and
  treatment, sterilization of water and sanitation.

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S. Typhi in the U.S.

• Almost 30 of reported cases in the U.S. are
  domestically acquired.
• Although most cases are sporadic, large outbreaks
  do occur.
• For example, outbreak linked to contaminated
  orange juice in N. Y.,
  caused by a previously unknown chronic carrier
  (1991).
• Multi-drug resistance
• recent trend toward an increased incidence of
  multi-drug resistant S. typhi in developing
  countries is reflected by increase in the
  proportion of U.S. cases 0.6 in 1985-1989 to
  1.2 in 1990-1994.

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Epidemiology

• S. enteriditis and typhimurium (non-typhoidal
  S.)
• - are the 2 top serotypes in the U.S. since
  1980s
• - cause gastroenteritis following ingestion of
  the bacteria on or in food or on fingers and
  other objects
• - cause the majority of cases of zoonotic
  salmonellosis in many countries.

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Salmonella Enteritidis

• Humpty Dumpty
• by R. Wayne Edwards January 1999
• Humpty Dumpty lay on the groundA crushed and
  broken fella.No one wanted to put him
  together'Cause he had salmonella.

• transmitted to humans by contaminated foods of
  animal origin, predominantly eggs. Raw eaten or
  undercooked eggs that have been infected in the
  hens ovaries can cause gastroenteritis

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Salmonella Enteritidis Infections, United States,
19851999

• During the 1980s, illness related to contaminated
  eggs occurred most frequently in the northeastern
  United States, but now it is increasing in other
  parts of the country as well.

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• CDC, 2002 In the Northeast, approximately one in
  10,000 eggs may be internally contaminated one
  in 50 average consumers could be exposed to a
  contaminated egg each year.
• In 1995 high of 3.9 per 100,000 population,
• In 1999 1.98 per 100,000, rate still
  decreasing due to prevention and control efforts
  by the government.

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S. typhimurium

• has been reported increasingly frequently as the
  cause of human and animal salmonellosis since
  1990, due to antibiotic resistance
• Predominant multi-drug resistant strain DT 104,
  which initially emerged in cattle in England,
  1988
• In 1997, the WHO stated that some countries in
  Europe had a staggering 20-fold increase in
  incidences between 1980 and 1997, and a 5-fold
  increase in the U.S. between 1974 and 1994, due
  to antibiotic resistant strains
• intensive animal maintenance.

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Epidemic measures

• Salmonellosis is a reportable disease.
• An intensive search should be conducted for
  the source of an infection and for the means
  (food or water) by which the infection was
  transmitted.
• Samples of blood can be taken immediately for
  confirmation and for testing for antibiotic
  sensitivity.
• Samples of stool or urine may be taken after
  one week of onset for effective confirmation.
• Food and water samples should be taken from
  suspected sources of the outbreak. It is
  recommended to organize temporary water
  purification and sanitation facilities until
  longer term measures can be implemented.

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Cost Estimates

• The cost per reported case of human salmonellosis
  range from US 100 to 1300 in North America and
  Europe.
• The costs associated with individual outbreaks in
  North America and Europe range from around
• 60,000 to more than 20 Million.
• The total annual cost in the U.S. is estimated a
  total of almost 400 Million.

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Salmonella

• Overview
• History and Epidemiology
• Molecular Biology
• Clinical
• Weaponization

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Salmonella Microbiology
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Classification

• Enterobacteria
• Gram-negative
• Facultative anaerobes
• Glucose-fermenting
• Straight, rod
• 2-3 µm in length
• Flagellated
• Many serovars
• Typhi
• Typhimurium
• Enteriditis

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LPS on Surface

• Lipopolysaccharide
• Protective outer layer of most strains
• (not S. typhi)
• Coded for by rfb locus on chromosome
• Lipid core of LPS highly conserved across
  serovars, but polysaccharide side chains are
  highly polymorphic (nature of rfb gene)

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LPS (cont.)

• Memory immune response and antibodies directed
  against LPS
• Polymorphic nature of side chains is advantageous
  for bacteria
• Since Typhi has outer capsule, this infection is
  worse.

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Infection

• Ingestion of contaminated food or water
• Passes through mucosa of intestine to epithelial
  cells
• Causes membrane ruffling
• Releases effector proteins through Type III
  Secretion system
• Endocytosis

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Salmonella Entry
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Membrane Ruffling
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Virulence Factors

• Genes for virulence factors cluster in
  pathogenicity islands (PI)
• Genes acquired through lateral transfer
• Bacteriophage and transposon insertion sequences
  flank PI
• Maybe vehicles for transfer of PI to Salmonella
  at one time
• Acquisition of PI enhances virulence of bacteria

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Horizontal Transfer

• Transformation
• Uptake of naked DNA
• Mediates exchange of any part of DNA
• Conjugation
• F to F-
• Requires cell to cell contact conjugation
  bridge
• Transduction
• Transfer of DNA by a phage
• New phage viral coat with bacterial DNA

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Salmonella Pathogenicity Islands

• Salmonella Pathogenicity Island 1 (SPI-1)
• entry into intestinal epithelium
• Enables pathogen to exploit host intestinal
  environment

• Salmonella Pathogenicity Island 2 (SPI-2)
• intracellular bacterial replication and
  initiation of systemic infection
• Do not influence enteropathogenesis to any great
  extent

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Type III Secretion System (TTSS)

• Main way Salmonella delivers virulence factors to
  host
• Made up of 20 proteins
• Assemble in step-wise order
• PrgI is a needle structure extended by protein
  base, forms a channel to host

PrgI
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Salmonella-host Interaction

• Two forms of TTSS
• One encoded on SPI-1, other on SPI-2
• SPI-1 TTSS probably causes initial interaction
• Starts bacteria-mediated endocytosis
• Entry activates SPI-2 TTSS to cause thorough
  infection

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Membrane Ruffling

• Cytoskeleton-associated proteins relocate to site
  of bacterial entry
• Bacterial effector proteins trigger cytoskeleton
  rearrangements
• Apical membrane surface undergoes structural
  changes, resembling ruffling
• This triggers endocytosis into vesicles
• Slightly different from receptor-mediated
  endocytosis

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Salmonella Containing Vesicle

• After ingestion, Salmonella enters a SCV through
  bacteria-mediated endocytosis
• Lives and multiplies in SCV
• Very little known about SCV or how bacteria exist
  inside
• A method to avoid host immune response
• Phagosome maturing SCV

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SPI-1 Effector Proteins

• SipA
• Binds actin and stabilizes filaments
• Allows actin to polymerize more easily
• Maximizes efficiency of Salmonella invasion

• SipC
• Aides in entry of other SPI-1 effector proteins
• Activtes G-actin to form F-actin, then polymerize
• Aides in cytoskeleton rearrangements in membrane
  ruffling

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SopB

• Main virulence factor
• Encoded by SPI-5
• An enterotoxin associated with SPI-1 TTSS
• Induces an increase in concentration of cellular
  inositol polyphosphate
• Increased chloride secretion into lumen
• Na follows to balance charge
• Water follow to balance osmolarity

diarrhea
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SPI-2 TTSS

• Activated once bacteria enters cell
• Necessary for systemic infection
• SPI-2 TTSS secretes effector proteins from
  phagosome into cytosol
• Interfere with maturation of phagosome
• No fusion with lysosome
• How Salmonella avoids degredation in cell

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Flagella

• Another antigen
• Host cytotoxic T-cell response directed against
  flagellar epitopes
• N- and C- termini are highly conserved
• Middle of flagellum is variable

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Phase I / II Flagella

• Operon encoding Phase I flagella also encodes for
  a protein that represses trascription of Phase II
• The switch mediated by an enzyme that inhibits
  Phase I, allowing Phase II
• May help Salmonella avoid cell-mediated immune
  response

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Tumor Necrosis Factor-a

• Flagella from S. Typhimurium induces expression
  of TNF-a through cell-mediated reponse
• Phase II flagella are less-potent inducers
• Switching mechanism may provide bacteria with a
  way to down-regulate inflammatory response within
  host

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Immune Response

• White blood cells recognize trigger T cells, B
  cells
• Two types of B cells one to attack now, one for
  memory
• Macrophages and neutrophils attack bacteria,
  secrete interleukins, causing cell-mediated
  response by T-cells
• Antibodies from B cells attach to bacteria,
  allowing cytotoxic T cells, macrophages, and
  neutrophils to kill the organism

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Inside Macrophages

• SPI-2 TTSS works in macrophages as well
• Bacterium produces enzymes that inactivte toxic
  macrophage compounds
• Homocysteine (Nitric Oxide antagonist)
• Superoxide dismutase (inactivates reactive
  peroxides)
• Salmonella must produce additional factors to
  survive limited nutrient base
• Allows bacteria to travel throughout body,
  causing systemic infection (only in S. typhi)

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Septicemia

• Invasion of bloodstream
• spv genes causes detachment of cells to ECM and
  apoptosis
• Spreads infection
• Bacteria can enter bloodstream and lymphatic
  system
• Main cause of death by Salmonella

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How do we respond?

• Microbiological view
• Vaccines
• Dam
• Antibiotics

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Salmonella Vaccine Strategy

• Delete chromosomal regions that code for
  independent and essential functions. This results
  in
• - low probability of acquiring both traits
• - both traits
• aro genes aromatic compound biosynthesis
• pur genes purine metabolism biosynthesis
• Deletion strains
• - can be grown on complete medium in lab
• - in vivo, growth is reduced
• - only a low level of infection is established
• - immune system can mount a response
• Vaccine suitable for humans and mice, chickens,
  sheep, cattle

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DNA adenine methylase (Dam)

• Enzyme that methylates specific adenine residues
  in Salmonella genome
• Disrupts regulation of DNA replication and repair
• Regulates expression of about 20 bacterial genes
  active during infection
• Dam (-) mutants are not virulent
• Good antimicrobial potential
• Current hot topic of research

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Antibiotics

• Antibiotics are selective poisons
• Do not harm body cells
• Target different aspects of bacteria, such as
  ability to synthesize cell wall, or metabolism
• MIC Minimum Inhibitory Concentration
• the minimum amount of agent needed to inhibit the
  growth of an organism

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Antibiotic Resistance

• Bacteria can counteract antibiotics by
• Preventing antibiotic from getting to target
• Changing the target
• Destroy the antibiotic
• Bacteria can acquire resistance
• Horizontal transfer from another bacteria
• Vertical transfer due to natural selection

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Salmonella

• Overview
• History and Epidemiology
• Molecular Biology
• Clinical
• Weaponization

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How Do You Catch Salmonella?

• Food borne
• Transmitted via improperly prepared, previously
  contaminated food or water
• - Meat poultry, wild birds, pork
• - Dairy eggs
• Pet turtles and lizards

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How does Salmonella affect the body?

• Three clinical forms of salmonellosis
• - Gastroenteritis (S. typhimurium)
• - Septicemia (S. Choleraesius)
• - Enteric Fevers (i.e. S. typhi Typhoid Fever)

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Who Can Be Infected?

• Everyone
• Especially the elderly, infants,
  immunocompromised patients (AIDS, sickle cell
  anemia)

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Factors Increasing Susceptibility
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Identification I

• Laboratory identification of genus Salmonella
  biochemical serological tests
• HOW?
• - stool or blood specimens are plated on agar
  media (bismuth sulfite, green agars, MacConkey)
• Suspect colonies further analyzed by triple sugar
  iron agar/ or lysine-iron agar
• - confirmed by antigenic analysis of O (somatic)
  and H (flagellar) antigens Test for antigens

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Identification II

• Use phenol red test
• - testing for lactic acid production
• - if negative, diagnose (presence of red
  spots surrounded by a bright red zone)

Salmonella typhimurium
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Nontyphoidal Salmonella

• General Incubation 6 hrs-10 days Duration 2-7
  days
• Infective Dose usually millions to billions of
  cells
• Transmission occurs via contaminated food and
  water
• Reservoir
• a) multiple animal reservoirs
• b) mainly from poultry and eggs (80 cases from
  eggs)
• c) fresh produce and exotic pets are also a
  source of contamination (gt 90 of reptile stool
  contain salmonella bacterium) small turtles ban.
  
• General Symptoms diarrhea with fever, abdominal
  cramps, nausea and sometimes vomiting

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Nontyphoidal Salmonella

• Caused by S. typhimurium and S. enteritidis
• Rainy season of tropical climates Warm season of
  temperate climates
• Growing rapidly in the U.S. five-fold increase
  between 1974-1994
• Centralization of food processing makes
  nontyphoidal salmonellosis particularly prevalent
  in developing countries
• Resistance is a concern, especially with
  multi-drug resistant S. Typhimurium known as
  Definitive Type 104 (DT 104)

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Nontyphoidal Salmonella Gastroenteritis

• Incubation 8-48 hrs Duration 3-7 days for
  diarrhea 72 hrs. for fever
• Inoculum large
• Limited to GI tract
• Symptoms include diarrhea, nausea, abdominal
  cramps and fevers of 100.5-102.2ºF. Also
  accompanied by loose, bloody stool
  Pseudoappendicitis (rare)
• Stool culture will remain positive for 4-5 weeks
• lt 1 will become carriers

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Nontyphoidal SalmonellaBacteremia and
Endovascular Infections

• 5 develop septicemia 5-10 of septicemia
  patients develop localized infections
• Endocarditis Salmonella often infect vascular
  sites preexisting heart valve disease risk
  factor
• Arteritis Elderly patients with a history of
  back/chest prolonged fever or abdominal pain
  proceeding gastroenteritis are particularly at
  risk.
• - Both are rare, but can cause complications
  that may lead
• to death

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Septicemia

• Serotype S. choleraesius causes septicemia
• - prolonged state of fever, chills, anorexia,
  and anemia
• - lesions in other tissues
• - septic chock, death

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Incidence of S. Enteritidis
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Nontyphoidal SalmonellosisLocalized Infections

• INTRAABDOMINAL INFECTIONS
• Rare, usually manifested as liver or spleen
  abscesses
• Risk factors hepatobiliary, abdominal
  abnormalities, sickle cell disease
• Treatment surgery to correct anatomic damages
  and drain abscesses
• CENTRAL NERVOUS SYSTEM INFECTIONS
• Usually meningitis (in neonates, present with
  severe symptoms e.g. seizures, hydrocephalous,
  mental retardation, paralysis) or cerebral
  abscesses
• PULMONARY INFECTIONS
• Usually lobar pneumonia
• Risk factors preexisting lung abnormalities,
  sickle cell disease, glucocorticoid usage

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Typhoidal Salmonellosis Enteric Fever

• Incubation 7-14 days after ingestion Duration
  several days
• Infective Dose 105 organisms
• Symptoms
• a) 1st week slowly increasing fever, headache,
  malaise, bronchitis
• b) 2nd week Apathy, Anorexia, confusion, stupor
• c) 3rd week rose spots (1-2 mm diameter on the
  skin) duration 2-5 days, variable GI symptoms,
  such as abdominal tenderness (majority),
  abdominal pain (20-40 of cases) and diarrhea
  enlargement of the spleen/liver, nose bleeds, and
  bradycardia
• neuropsychiatric symptoms delirium and mental
  confusion
• Long term effects arthritis

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Typhoidal Salmonellosis

• Late stage complications include intestinal
  perforation and gastrointestinal hemorrhage
• Immediate care such as increase antibacterial
  medications or surgical resection of bowel
• Other rare complications include inflammation of
  the pancreas, endocardium, perocardium,
  myocardium, testes, liver, meninges, kidneys,
  joints, bones, lungs and parotid gland and
  hepatic/splenic abscesses
• In general, symptoms of paratyphoid fever are
  similar to typhoid fever, but milder with a lower
  mortality rate
• Majority of bacteria gone from stool in 8 weeks
  However, 1-5 become asymptomatic chronic
  carriers gallbladder is the primary source of
  bacterium

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Typhoidal Salmonella
Chest PA view shows pleural effusion, left lower
pulmonary lobe atelectasis, medial and downward
shift of bowel gas, and an increase in the
air-fluid level in the abdomen
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Pictures
(A)
(B)
(A) In sub-acute infections, multiple white to
yellow foci occur in the liver, spleen is
enlarged, and mesenteric lymph nodes may be
enlarged (B) Histopathological examination may
reveal necrotizing splenitis and hepatitis, with
necrotic foci often accompanied by colonies of
bacteria (arrow in right photo).
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Treatment of Typhoidal Salmonellosis

• Third generation cephalosporins or quinolones is
  the current treatment
• IV or IM ceftriaxone (1-2g) is also prescribed
  usually 10-14 days (5-7 days for uncomplicated
  cases)
• Multi Drug Resistant (MDR) strains of S. typhi
  quinolones are the only effective oral treatment
• Nalidixic acid resistant S. typhi (NARST) must be
  tested for sensitivity to determine course of
  treatment
• Sever typhoid fever (altered consciousness,
  septic shock) dexamethasone treatment
• Chronic carriers 6 weeks of treatment with
  either oral amoxicillin, ciprofloxacin,
  norfloxacin
• Surgical intervention to remove damaged cells

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Prevention

• Typhoidal S.
• - Generally treated with antibiotics
• - vaccinations available the CDC currently
  recommends vaccination for persons traveling to
  developing countries
• - Education of general public, especially in
  developing countries identification of all
  carriers and sources of contamination of water
  supplies
• - avoid risky foods drinks
• buy bottled water or boil water for at least
  1 minute
• COOK and CLEAN food thoroughly, avoid raw
  vegetables and fruits
• - WASH YOUR HANDS WITH SOAP AND WATER!!!

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• Preventive measures for non-typhoidal S.
• - pasteurization of milk-products Eggs from
  known infected commercial flocks will be
  pasteurized instead of being sold as grade A
  shell eggs.
• - tracebacks, on-farm testing, quality assurance
  programs, regulations regarding refrigeration,
  educational messages for safe handling and
  cooking of eggs
• - Cross-contamination uncooked contaminated
  foods kept separate from cooked, ready-to-eat
  foods.

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Salmonella Vaccines I

• Poultry vaccine MeganVac 1
• - applied to baby chicks via drinking water and
  cattle. It stimulates immunity in the chickens,
  preventing Salmonella infection during the
  growing period which may result in contamination
  and subsequent food borne infection of humans
• - targets S. Enteritidis
• - Salmonella infection is stopped at lower
  levels of the food chain will mean increased
  productivity for the farmer and a break in the
  cycle of Salmonella transmission from animals à
  humans

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Salmonella Vaccines II

• Today, three types of Typhoid Vaccines are
  available
• (1) inactivated whole-cell vaccine 2 doses/
  4wks. Apart single booster dose recommended
  every 3 years
• (2) Ty21a a live, attenuated S. typhi vaccine.
  Administered orally (4 doses). Efficacy 7 years
• (3) Vi polysaccharide vaccine from purified Vi
  polysaccharide from S. typhi. Administered
  subcutaneously or intramuscularly. To maintain
  protection, revaccination recommended every 3
  years.
• These vaccines have been shown be 70-90
  effective.

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Salmonella

• Overview
• History and Epidemiology
• Molecular Biology
• Clinical
• Weaponization

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Salmonella as A Bioweapon?
107
CDC classification

• Category B agent includes microorganisms that
  are moderately easy to disseminate, have moderate
  morbidity (i.e., ability to cause disease) and
  low mortality, but require enhanced disease
  surveillance.
• Biosafety Level 2
• Risk Level 2 associated with human disease that
  is rarely serious and prophylactic intervention
  is often available.
• 9 different species Salmonella arizonae,
  cholerasuis, enteritidis, gallinarum-pullorum,
  meleagridis, paratyphi (Type A,B,C), spp., typhi,
  and typhimurium
• Salmonella typhi is the only species that
  requires import and/or export permit from CDC
  and/or Department of Commerce has high droplet
  or aerosol production potential

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WHO Global Salm Surv (GSS)

• GSS is an international Salmonella surveillance
  program initiated in January 2002. It collects
  annual summary data from member institutions all
  over the world.
• The goal is to enhance the quality of Salmonella
  surveillance, serotyping and antimicrobial
  resistance testing and leading local
  interventions that reduce the human health burden
  of Salmonella.
• A total of 138 laboratories were enrolled in the
  GSS in September 2003.

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Salmonella as a Bioterrorist Weapon What states
are most at risk?

• The states most vulnerable to terrorist attack on
  the agricultural sector are those with several or
  most of the following attributes
• High density, large agricultural area
• heavy reliance on monoculture of a restricted
  range of genotypes
• major agricultural exporter, or heavily dependent
  on a few domestic agricultural products
• suffering serious domestic unrest, or the target
  of international terrorism, or unfriendly
  neighbor of states likely to be developing BW
  programs

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First Use of Salmonella as a Bioterrorist Weapon

• From 1932-1945, Japan conducted biological
  warfare experiments in Manchuria
• At Unit 731, a biological warfare research
  facility, prisoners were infected with Salmonella
  typhosa among other biological agents
• Additionally, a number of Chinese cities were
  attacked. The Japanese contaminated water
  supplies and food items with Salmonella. Cultures
  were also tossed into homes and sprayed from
  aircraft
• Due to inadequate preparation, training, and/or
  lack of proper equipment, the Chekiang Campaign
  in 1942 led to about 10,000 biological casualties
  and 1,700 deaths among the Japanese troops.

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• Oregon 1984 a religious cult known as the
  Rajneeshees, a Buddhist cult sought to run
• the whole country by wining the local election in
  1984 using salmonella bacteria. They
• brewed a "salsa" of salmonella and sprinkled it
  on the town's restaurant salad bars. Ten
• restaurants were hit and more than 700 people got
  sick.
• First large scale bioterrorism attack on
  American soil
• A communitywide outbreak of salmonellosis
  resulted at least 751 cases were
• documented in a county that typically reports
  fewer than five cases per year.
• Health officials soon pinned down salmonella as
  the cause of the sudden outbreak, but
• put the blame on food handlers. In 1984, who
  could have imagined bioterrorism?  
• caused by S. typhimurium as this type

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Salmonella as a Bioterrorist Weapon

• Wide distribution of food contaminated food
  produced in one country can cause illness in
  other countries
• Traceability
• Antimicrobial resistance strains of
• Salmonella are being found that have
• multiple drug resistance
• Capacity building Salm-gene project
• used to enhance outbreak detection by
• routinely sub-typing certain salmonellas
• using molecular methods

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Salmonella as a Bioterrorist Weapon

• Contaminating unguarded food supplies
• Some terrorist acts may be designed purely to
  spread panic contaminating the food supply could
  bring economic and agricultural production to a
  standstill
• EX. If numerous food-borne outbreaks occurred
  across the country, people would soon fear their
  meals
• Unfortunately, people have reason to worry all
  these contaminations have occurred naturally
  every year. If Mother Nature can do this
  repeatedly, then a terrorist should have no
  problem recreating these outbreaks over and over
  in any number of American cities.

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Salmonella as a Bioterrorist Weapon

• readily accessible and easy to grow or make
• Centralized food production largely unmonitored
  food supply food that is tampered with can be
  widely quickly distributed
• Terrorist groups could use infectious disease
  agents to confuse public health officials into
  believing that outbreaks are naturally occurring
  it is estimated that 1.4 million salmonella
  infections occur each year, but the CDC gets
  reports of only about 38,000 annually
• According to the Centers for Disease Control
  (CDC), only 32 of the reported outbreaks have a
  known etiology.

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Salmonella as a Bioterrorist Weapon

• No food product is safe vegetables and fruits
  are the easiest to contaminate. Fresh-produce
  wholesalers and distributors are notorious for
  employing illegal immigrants and not checking
  their background information.
• Even processed foods arent safe Terrorists
  could use heat-stable toxins that would survive
  the packaging process.
• As more of our food becomes imported, especially
  hard-to-clean off-season fruits and vegetables,
  bioterrorists dont even have to be inside the
  United States to do damage

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Salmonella as a Bioterrorist Weapon Who might be
tempted to initiate an attack on the agricultural
sector?

• Terrorist groups might be interested in
  agricultural bioweapons for a variety of reasons
  
• 1. international terrorist organizations cause
  harm/injury to enemy states or peoples
• - in an ideologically-motivated terrorist
  attack there would be willing assumption of
  responsibility by the perpetrator OR an attempt
  to disguise the outbreak as natural.
• 2. Extreme activist groups
• - EX. anti-GMO groups for their potential value
  in deterring farmers from the use of genetically
  engineered crops or animals

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Salmonella as a Bioterrorist Weapon What goals
might an attack on the agricultural sector serve?

• Food attack by a terrorist group initiate
  point-source epidemics using available Salmonella
  strains
• Destabilize a government by initiating food
  shortages/unemployment the potential for immense
  economic damage due to contamination of the food
  supply
• Alter supply and demand patterns for a commodity
  an outbreak can trigger the imposition of trade
  restrictions. This is turn would open up or close
  markets for others.

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Salmonella as a Bioterrorist Weapon What are the
special features of an attack on the agricultural
sector?

• Salmonella is not hazardous to perpetrators
  easy to produce, stockpile, and disseminate
• Few technical obstacles to weaponization it
  would not be difficult to obtain Salmonella
  strains on the open market.
• Low security of vulnerable targets Fields,
  supermarkets, restaurants have essentially no
  security at all.
• Point source to mimic natural introduction
  Because of the high incidence of
  naturally-occurring diseases, a deliberately
  instigated outbreak could be mistaken for a
  natural one
• Multiple point source outbreaks can be initiated
  by contaminating imported feed or fertilizer,
  without even entering the country allows the
  possibility of initiating multiple outbreaks over
  a large geographic area, in a way that mimics a
  natural event

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Salmonella Dilemma

• Dissemination of genomic knowledge of salmonella
  can facilitate bio-weapons development
• Alternative 1 Restrict dissemination of genomic
  knowledge
• - short term hinders development of a
  super-Salmonella terror weapon
• - long run leaves us at the mercy of multi-drug
  resistant salmonella strains ranging from
  incapacitating to lethal
• Alternative 2 Disseminate genomic knowledge, but
  support development of salmonella specific-drugs
• - knowledge may provide a terrorist org. with
  the ability to develop super-Salmonella terror
  weapons, but it provides us with the opportunity
  to defend against all salmonella infection.

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Combating Salmonella Bioterrorism

• Establish a national disease surveillance system
  that could not only help uncover a terrorist
  attack but also recognize naturally occurring
  outbreaks that now go undetected
• New technology creating a diagnostic gene chip
  covering all major diseases could give the health
  care provider instant diagnoses. Similar gene
  chips could monitor the health of livestock,
  poultry, and crops. Chips could be used during
  various steps of food processing to ensure
  quality control and food safety.

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Lines of Defense

• Food processors should limit access to their
  production, storage and packaging areas
  rerouting traffic, installing locks
• Randomized safety checkpoints will increase fear
  of detection
• COSTS
• Increase work force
• Sampling and test costs
• Record keeping

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Government Action

• CDC monitors the frequency of Salmonella
  infections in the country and assists the local
  and State Health Departments to investigate
  outbreaks and devise control measures
• FDA inspects imported foods, milk pasteurization
  plants, promotes better food preparation
  techniques in restaurants and food processing
  plants, and regulates the sale of turtles and it
  also regulates the use of specific antibiotics as
  growth promotants in food animals
• USDA monitors the health of food animals,
  inspects egg pasteurization plants, and is
  responsible for the quality of slaughtered and
  processed meat.
• EPA regulates and monitors the safety of our
  drinking water supplies.

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Biological Weapon Prevention

• BTWC (Biological and Toxin Weapons Convention)
  drafted in 1972
• - intended to prevent the development,
  production and stockpiling of biological weapons
• - pathogens or toxins in quantities that have no
  justification for protective or peaceful services
  are to be eliminated
• - today, 159 countries have signed the
  convention and 141 have ratified it
• - however, more can be done Factories in the
  former Eastern Europe supply viruses that cause
  fatal diseases, such as E-Coli and Salmonella,
  without checking the identities of the
  purchasers (from the trials of the largest
  fundamentalist org. in Egypt, Abu-al-Dahab)

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Acknowledgements

• Dr. Geoffrey Zubay
• Salwa Touma
• Kathleen Kehoe

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