Salmonella Praveen Rao Sophia W Riccardi Danielle Birrer

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

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

Overview • Salmonella is a rodshaped, gramnegative, facultative anaerobe in the family Enterobacteriaceae

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.

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 -60°C or 134 -140°F has shown to be effective in killing the bacteria. Optimal growth: 37°C or 98. 6°F •

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.

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) ٠ causes typhoid and paratyphoid fever ٠ restricted to growth in human hosts ٠ principal habitat is in intestinal tracts and the bloodstream • • •

- Nontyphoidal Salmonella (example: S. enteritidis, S. typhimurium) ٠ prevalent in gastrointestinal tracts of a broad range of animals, including mammals, reptiles, birds and insects. ٠ cause a whole range of diseases in animals and humans, mainly gastroenteritis. ٠ usually transferred animal-to-person, through certain food products: fresh meat, poultry, eggs and milk - fruits, vegetables, seafood ٠ house and exotic pets, contamination through contact with their feces

- 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.

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

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 Alexander’s 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.

History l 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. l

History Typhoid outbreak in British camps during the South African War (18991902) - 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 washed into the network of streams and rivers during the rainy season. l l Epidemic potential during a war prominent because of the disposal problems of men’s discharges.

History l 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.

Salmonella vaccine l First preventive measure against Salmonella was discovered in 1896, as an antityphoid vaccine was developed by the British surgeon Almroth Wright. l 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 l l 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”.

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).

First typhoid inoculation, 1909 United States Army Medical School Bottling typhoid vaccine, 1944 Division of Biologic Products, U. S. Army of Medical Department Professional Service Schools

History in the U. S. l “Typhoid Mary” Mallon was the first famous carrier of typhoid fever in the U. S. l 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 nontyphoidal 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 1900’s. l l

History: Mary Mallon l 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. l 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. l 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.

Recent outbreaks More recently reported outbreaks in the U. S. involve different kinds of Salmonella strains, predominantly S. enteritidis and S. typhimurium. l 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. l

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

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).

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) •

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.

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.

Relative Frequency of the disease in the U. S. l 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. l

Epidemiology The most commonly reported serotypes, in history and present: - S. typhi - S. enteritides and S. typhimurium l l The “top 20” serotypes accounted for 80% of all isolates reported in the U. S. in 2001.

Top 15 Salmonella Serotype list in the U. S. , 2001 Country, Institution, Biological origin Total Serot ped U. S. A. , Centers of Disease Control, Control and Prevention-FDDB Epi, 2001, Human 31, 675 Rank Serotype % of Total Serotyped Count 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

Epidemiology S. typhi (typhoidal Samonella) l Causes enteric fever l Have no known hosts other than humans. l 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. l 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.

l l 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.

Salmonella typhi in developing countries l l 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.

S. Typhi in the U. S. l 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). l 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. l

Epidemiology l S. enteriditis and typhimurium (non-typhoidal S. ): - are the 2 top serotypes in the U. S. since 1980’s - 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.

Humpty Dumpty Salmonella Enteritidis l transmitted to humans by contaminated foods of animal origin, predominantly eggs. Raw eaten or undercooked eggs that have been infected in the hen’s ovaries can cause gastroenteritis by R. Wayne Edwards January 1999 Humpty Dumpty lay on the ground A crushed and broken fella. No one wanted to put him together 'Cause he had salmonella.

l During the 1980 s, 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. Salmonella Enteritidis Infections, United States, 1985– 1999

l 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. l

S. typhimurium l has been reported increasingly frequently as the cause of human and animal salmonellosis since 1990, due to antibiotic resistance l Predominant multi-drug resistant strain DT 104, which initially emerged in cattle in England, 1988 l 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 l intensive animal maintenance.

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. l 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. l l 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.

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

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

Salmonella Microbiology

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

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)

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.

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

Salmonella Entry

Membrane Ruffling

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

Horizontal Transfer • Transformation • • • Conjugation • • • Uptake of naked DNA Mediates exchange of any part of DNA F+ to FRequires cell to cell contact – conjugation bridge Transduction • • Transfer of DNA by a phage New phage: viral coat with bacterial DNA

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

Type III Secretion System (TTSS) • • Main way Salmonella delivers virulence factors to host Made up of 20 proteins • • Assemble in step-wise order Prg. I is a needle structure extended by protein base, forms a channel to host Prg. I

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

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

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

SPI-1 Effector Proteins • Sip. A • • • Binds actin and stabilizes filaments Allows actin to polymerize more easily Maximizes efficiency of Salmonella invasion • Sip. C • • • 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

Sop. B • • 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

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

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

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

Tumor Necrosis Factor-α • • • Flagella from S. Typhimurium induces expression of TNF-α 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

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 Tcells Antibodies from B cells attach to bacteria, allowing cytotoxic T cells, macrophages, and neutrophils to kill the organism

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)

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

How do we respond? • Microbiological view • • • Vaccines Dam Antibiotics

Salmonella Vaccine Strategy l l l 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

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

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

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

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

How Do You Catch Salmonella? Food borne l Transmitted via improperly prepared, previously contaminated food or water - Meat: poultry, wild birds, pork - Dairy: eggs l Pet turtles and lizards l

How does Salmonella affect the body? Three clinical forms of salmonellosis l - Gastroenteritis (S. typhimurium) l - Septicemia (S. Choleraesius) l - Enteric Fevers (i. e. S. typhi – Typhoid Fever) l

Who Can Be Infected? Everyone l Especially: the elderly, infants, immunocompromised patients (AIDS, sickle cell anemia) l

Factors Increasing Susceptibility

Identification I l l l Laboratory identification of genus Salmonella: biochemical + serological tests HOW? - stool or blood specimens are plated on agar media (bismuth sulfite, green agars, Mac. Conkey) 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:

Identification II Use phenol red test: - testing for lactic acid production - if negative, diagnose (presence of red spots surrounded by a bright red zone) l Salmonella typhimurium

Nontyphoidal Salmonella l l l 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 (> 90% of reptile stool contain salmonella bacterium); small turtles ban. General Symptoms: diarrhea with fever, abdominal cramps, nausea and sometimes vomiting

Nontyphoidal Salmonella l l l 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)

Nontyphoidal Salmonella: Gastroenteritis l l l 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 < 1% will become carriers

Nontyphoidal Salmonella: Bacteremia and Endovascular Infections l l l 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

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

Incidence of S. Enteritidis

Nontyphoidal Salmonellosis: Localized Infections l l l l l 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

Typhoidal Salmonellosis: Enteric Fever l l l Incubation: 7 -14 days after ingestion; Duration: several days Infective Dose = 105 organisms Symptoms: a) 1 st week: slowly increasing fever, headache, malaise, bronchitis b) 2 nd week: Apathy, Anorexia, confusion, stupor c) 3 rd 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

Typhoidal Salmonellosis l l l 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 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

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

Pictures (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). (B)

Treatment of Typhoidal Salmonellosis l l l l Third generation cephalosporins or quinolones is the current treatment IV or IM ceftriaxone (1 -2 g) 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

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!!! l

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.

Salmonella Vaccines I l Poultry vaccine: Megan™Vac 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

Salmonella Vaccines II l Today, three types of Typhoid Vaccines are available: (1) inactivated whole-cell vaccine: 2 doses/ 4 wks. Apart; single booster dose recommended every 3 years (2) Ty 21 a: 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. l These vaccines have been shown be 70 -90% effective.

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

CDC classification l l l 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

WHO Global Salm Surv (GSS) l GSS is an international Salmonella surveillance program initiated in January 2002. It collects annual summary data from member institutions all over the world. l 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. l A total of 138 laboratories were enrolled in the GSS in September 2003.

Salmonella as a Bioterrorist Weapon: What states are most at risk? l The states most vulnerable to terrorist attack on the agricultural sector are those with several or most of the following attributes: l 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 l l l

First Use of Salmonella as a Bioterrorist Weapon l l 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.

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

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

Salmonella as a Bioterrorist Weapon l l 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 l 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.

Salmonella as a Bioterrorist Weapon l l 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.

Salmonella as a Bioterrorist Weapon l 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. l Even processed foods aren’t safe: Terrorists could use heat-stable toxins that would survive the packaging process. l As more of our food becomes imported, especially hard-to-clean off-season fruits and vegetables, bioterrorists don’t even have to be inside the United States to do damage

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

Salmonella as a Bioterrorist Weapon: What goals might an attack on the agricultural sector serve? l Food attack by a terrorist group: initiate point-source epidemics using available Salmonella strains l Destabilize a government by initiating food shortages/unemployment: the potential for immense economic damage due to contamination of the food supply l 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.

Salmonella as a Bioterrorist Weapon: What are the special features of an attack on the agricultural sector? l Salmonella is not hazardous to perpetrators: easy to produce, stockpile, and disseminate l Few technical obstacles to weaponization: it would not be difficult to obtain Salmonella strains on the open market. l Low security of vulnerable targets: Fields, supermarkets, restaurants have essentially no security at all. l 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 l 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

Salmonella Dilemma l l l Dissemination of genomic knowledge of salmonella can facilitate bioweapons 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.

Combating Salmonella Bioterrorism l 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 l 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.

Lines of Defense l l l 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

Government Action l l 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.

Biological Weapon Prevention l 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)

Acknowledgements • • • Dr. Geoffrey Zubay Salwa Touma Kathleen Kehoe