Shigella

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Shigella :

Shigella 1/22/2013 1 Shigella

HISTORY - 1888:

HISTORY - 1888 Andre Chantemesse Georges- Fernand - Isidor Widal First to recognize 1/22/2013 2 Shigella French bacteriologist French physician

HISTORY……:

HISTORY…… Kiyoshi Shiga Isolated and identified in 1898 Shigella dysenteriae 1 1/22/2013 3 Shigella Japanese microbiologist

HISTORY……:

HISTORY…… The genus name Shigella  1919 by Castellani and Chalmers in the honour of its discoverer Aldo Castellani 1/22/2013 4 Shigella Florence bacteriologist

Shigella :

Shigella Scientific classification Kingdom : Bacteria Phylum : Proteobacteria Class : Gamma Proteobacteria Order : Enterobacteriales Family : Enterobacteriaceae Genus : Shigella 1/22/2013 Shigella 5 Magnification: x2,200

Genus and species definition :

Genus and species definition Fermentative, facultatively anaerobic, oxidase negative, non citrate utilizing, non spore-forming, non capsulated, gram-negative, non motile rods Do not produce gas from carbohydrate Form acetylmethylcarbinol decarboxylate lysine hydrolyze arginine 1/22/2013 6 Shigella

Species :

Species Four Shigella dysenteriae - A Shigella flexneri - B Shigella boydii - C Shigella sonnei - D Differ by biochemical and serological characters. The G + C content of the Shigella is 48–53 mol% 1/22/2013 7 Shigella

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A physician, scientist, administrator, & professor of experimental Pathology at the University of Pennsylvania Described Sh flexeneri from Philippines in 1900 1/22/2013 Shigella 8 Simon Flexner, M.D

Shigella boydii:

Shigella boydii Mark Frederick Boyd an American Bacteriologist  discovered Shigella boydii in 1931 from India 1/22/2013 Shigella 9

Shigella sonnei:

Shigella sonnei Carl Olaf Sonne a Danish bacteriologist and parasitologist  discovered Shigella sonnei in 1915 1/22/2013 Shigella 10

Shigella:

Shigella Parasite of the digestive tract of humans Infective dose is small Produce enterotoxin Cause shigellosis  mild diarrhoea / bacillary dysentery 1/22/2013 11 Shigella

Antigenic structure:

Antigenic structure Complex 13 serotypes of Sh. dysenteriae  type 1 cause severe bacillary dysentery Sh. boydii  18 serotypes Sh. flexneri  six serotypes  subdivided into subtypes Sh. sonnei are serologically homogenous 1/22/2013 12 Shigella

Habitat & transmission:

Habitat & transmission Humans  reservoir of Shigella Monkeys  carriers Faeco –oral transmission Transported  flies 1/22/2013 13 Shigella

Clinical manifestations :

Clinical manifestations Shigellosis  acute recto-colitis Invasion of the rectal and colonic epithelia by Shigella Damage is mostly recto-sigmoid area  mucosa appearing swollen, haemorrhages, and ulcers 1/22/2013 14 Shigella

Clinical manifestations :

Clinical manifestations Onset sudden Initial symptom abdominal colic & cramps Watery diarrhoea  bloody mucus Fever, headache, malaise and anorexia 1/22/2013 15 Shigella

Clinical manifestations:

Clinical manifestations Symptoms last about 4 -14 days or more Can affect CNS  seizures and encephalitis Shigellosis complications  Reiter's syndrome  expression of HLA-B 27 histocompatibility antigen HUS 1/22/2013 16 Shigella

Clinical manifestations:

Clinical manifestations The severity  species involved Sh. dysenteriae 1  severe Sh. flexneri and Sh. boydii groups also  severe illness Sh. sonnei ( Sonnei dysentery )  few loose stools with vague abdominal discomfort 1/22/2013 17 Shigella

Clinical manifestations.....:

Clinical manifestations..... Strains of Sh. dysenteriae 1  haemolytic uraemic syndrome It is associated with complement activation and DIC in children Death from bacillary dysentery  extremes of life or with debilitating condition 1/22/2013 18 Shigella

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AT RISK GROUPS Children in day care centers International travelers Patients with HIV infection People living with inadequate water supply Persons in prisons & military camps 1/22/2013 19 Shigella

Distribution of serogroups :

Distribution of serogroups S.dysenteriae  serious disease South Asia Sub Saharan Africa S. flexneri  most commonly isolated in developing countries  1b, 2a,3a,4a,& 6 S.sonnei  mild form of diarrhea  endemic S.boydii  rare 1/22/2013 20 Shigella

Genome structure :

Genome structure Shigella dysenteriae has the smallest genome of the genus Shigella Its genome consists of a single circular chromosome and 4.37 mbp 2.12 mbp virulence plasmid 1/22/2013 Shigella 21

Pathogenesis of shigellosis :

Pathogenesis of shigellosis Shigella transit through gastric barrier and small intestine Reaching site of invasion  colonic and rectal mucosa 1/22/2013 22 Shigella

Pathogenesis....:

Pathogenesis.... Shigella cannot invade epithelial cells Exploits M cells of the follicle-associated epithelium  poorly organized brush border 1/22/2013 23 Shigella

Pathogenesis.....:

Pathogenesis..... Bacteria reach  area of the lymphoid follicle  phagocytosed by macrophages and dendritic cells Shigella kills macrophages by apoptosis Escapes to the sub-epithelial tissue Invades the epithelial lining baso -laterally 1/22/2013 24 Shigella

Pathogenesis.....:

Pathogenesis..... Polymorphonuclear cells (PMN) infiltrate Bacteria  disseminate throughout the epithelium by spreading from cell to cell Express pro-inflammatory cytokines and chemokines  IL-8  exacerbating inflammation 1/22/2013 25 Shigella

Pathogenesis.....:

Pathogenesis..... Occurrence of an acute rectocolitis with epithelial abscesses and ulcerations Spreading process  strong inflammatory reaction  rupture and destruction of the epithelial barrier 1/22/2013 26 Shigella

Pathogenesis:

Pathogenesis 1/22/2013 27 Shigella

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28 1/22/2013 Shigella

Pathology:

Pathology Rectosigmoidal lesions of shigellosis resemble those of ulcerative colitis Proximal extension of erythema , edema , loss of vascular pattern, focal hemorrhage , and adherent layers of purulent exudate 1/22/2013 Shigella 29

Pathology......:

Pathology...... Biopsy specimens typically shows oedema, with capillary congestion focal haemorrhage crypt hyperplasia goblet cell depletion mononuclear and polymorphonuclear (PMN) cell infiltration shedding of epithelial cells and erythrocytes Micro ulcerations 1/22/2013 Shigella 30

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Virulence in shigella species is determined by chromosomal & plasmid-coded genes VIRULENCE Shigella invades colonic mucosa & causes cell necrosis Chromosomal genes control cell wall antigens that are resistant to host defense mechanisms Plasmid genes control production of cytotoxin  enterotoxin and neurotoxin 1/22/2013 32 Shigella

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Lipopolysaccharide  preventing the effects of serum complement Lipid A component  localized cytokine release  inflammatory response & cellular disruption enable entry Sh. flexneri and Sh. sonnei express an aerobactin -mediated high-affinity iron uptake system Sh. dysenteriae 1 does not express this siderophore 1/22/2013 Shigella 33 VIRULENCE

Plasmid :

Plasmid The 213 kb virulence plasmid contains the genes required to express the Shigella invasive phenotype The presence of this plasmid was discovered in 1980s 1/22/2013 34 Shigella

Functions of plasmid:

Functions of plasmid 1]Adhesins 2]Invasion plasmid antigens ( Ipa ) that have a direct role in the Shigella invasion process 3]Transport or processing functions  ensure the correct surface expression of the Ipa proteins 1/22/2013 Shigella 35

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Ipa proteins are expressed in intestinal lumen (bile salts, high osmolarity , and human body temperature) This induces the endocytic uptake of shigellae by M cells, epithelial cells, and macrophages Ipab also mediates lysis of endocytic vacuoles in epithelial cells or macrophages. 1/22/2013 Shigella 36

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Ipa proteins also cause release of the IL-1 cytokine and macrophage apoptosis Plasmid-encoded virulence determinant is Intercellular Spread ( IcsA ) protein  polymerization of filamentous actin 1/22/2013 Shigella 37

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Formation of this actin tail provides a motive force for shigellae The Icsb plasmid-encoded protein then lyses the plasma membranes  intercellular bacterial spread 1/22/2013 Shigella 38

Functions of plasmid…..:

Functions of plasmid….. 4]The induction of endocytic uptake of bacteria and disruption of endocytic vacuoles 5]The intra- and inter-cellular spreading phenotypes 6]The regulation of plasmid-encoded vir genes 1/22/2013 Shigella 39

The Shiga Toxin :

The Shiga Toxin Expression of Shiga toxin has been shown to be iron regulated  toxin production increasing under conditions of iron restriction The toxin has a molecular weight of 68,000 da A subunit  responsible for the toxic action of the protein B subunit  responsible for binding to a specific cell type globotriosylceramide Gb 3 1/22/2013 Shigella 40

The Shiga Toxin :

The Shiga Toxin Shiga toxin becomes internalized by host cells  active within endosomes  Golgi apparatus A subunit divides to form portions A 1 and A 2 A 1 portion of the toxin prevents protein synthesis and causes cell death  Cytotoxic effect 1/22/2013 Shigella 41

The Shiga Toxin :

The Shiga Toxin Haemolytic uraemic syndrome  Shiga toxin on kidney tissues The prototype of Shiga toxin is found in S. dysenteriae serotype 1  Stx Subtypes  Stx1, Stx1c, Stx2, etc 1/22/2013 42 Shigella

The Shiga Toxin :

The Shiga Toxin Source Organism Gene Designation Toxin Name Older Names Shigella dysenteriae , type 1 stx Shiga toxin (Stx) Shiga toxin Escherichia coli stx1 Shiga toxin 1 (Stx1) Shiga-like toxin I, Verotoxin 1 stx2 Shiga toxin 2 (Stx2) Shiga-like toxin II, Verotoxin 2 1/22/2013 Shigella 43

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Stx damages intestinal capillaries  killing the cells of the blood vessels Stx triggers neutrophil -rich inflammation reactions  further damage epithelial layers A specific target for the toxin  vascular endothelium of the glomerulus  development of hemolytic uremic syndrome 1/22/2013 44 Shigella

Other effects of Shiga toxin:

Other effects of Shiga toxin Enterotoxic effect Neurotoxic effect 1/22/2013 45 Shigella

Enterotoxic effect:

Enterotoxic effect Toxin binds to receptors on intestinal epithelial cells  blocks absorption of electrolytes and nutrients from the intestinal lumen This leads Diarrhoea Dysentery Dehydration Loss of electrolytes Impaired nutrient absorption 1/22/2013 Shigella 46

Neurotoxic effect :

Neurotoxic effect CNS abnormalities include Lethargy Disorientation Seizures Paralysis Coma Fever and abdominal cramps are considered signs of neurotoxicity 1/22/2013 47 Shigella

Host Defence:

Host Defence Can cause disease after the ingestion of as few as 10 organisms Shigellosis an acute, self-limiting disease  regenerative capacity of the intestinal epithelium 1/22/2013 Shigella 48

Host Defence.....:

Host Defence..... Tenesmus and evacuation of mucus  effectively eliminate both extracellular shigellae and infected enterocytes This defensive response  constitutes the definitive sign of bacillary dysentery 1/22/2013 Shigella 49

Host Defence:

Host Defence Shigellosis childhood disease The incidence decreases drastically in the indigenous population over 5 years of age Studies in North American adults indicate prior infection with Sh. flexneri protects against reinfection (70% efficacy) 1/22/2013 Shigella 50

Lab diagnosis:

Lab diagnosis Feacal leucocytes Isolation Typing ELISA Molecular diagnosis Vero and HeLa cell tests 1/22/2013 Shigella 51

Laboratory diagnosis.....:

Laboratory diagnosis..... Shigellae in stools 10 3 – 10 9 cfu per gram Extremely fragile and they quickly die off Can survive in buffered glycerol solution / at 4 0 C 1/22/2013 52 Shigella

Laboratory diagnosis.....:

Laboratory diagnosis..... Specimens should be immediately inoculated into cultured media Plating on isolation medium should ideally be carried out within 6 h 1/22/2013 53 Shigella

Microbiological examination :

Microbiological examination A specimen of faeces is preferable Inoculated on desoxycholate citrate agar or MacConkey agar 1/22/2013 54 Shigella

Desoxycholate citrate agar:

Desoxycholate citrate agar Peptone 5g/l Lactose 10g/l Sodium citrate 8.5g/l Sodium thiosulfate 5.4g/l Ferric citrate 1g/l Sodium deoxycholate 5g/l Neutral red 20 mg/l Agar 15g/l 1/22/2013 55 Shigella

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Non-lactose / late lactose fermenting colonies are tested by standard biochemical and sugar utilization tests Identity is confirmed by agglutination tests with species-specific antisera , and then with type-specific sera 1/22/2013 56 Shigella

Isolation of Shigella :

Isolation of Shigella Hektoen , Salmonella- Shigella (SS), or xylose -lysine- deoxycholate (XLD) agar MacConkey or eosin-methylene blue (EMB) agar Bromocresol -purple agar lactose (BCP) 1/22/2013 57 Shigella

Hektoen agar :

Hektoen agar 1/22/2013 58 Shigella

Hektoen agar :

Hektoen agar Enzymatic Digest of Animal Tissue 16.5 g Yeast Extract 3 g Bile Salts 4.5 g Lactose 12 g Sucrose 12 g Salicin 2 g Sodium Chloride 5 g Sodium Thiosulfate 5 g Ferric Ammonium Citrate 1.5 g Bromthymol Blue 0.065 g Acid Fuchsin 0.1 g Agar 13.5 g Final pH: 7.6 ± 0.2 at 25°C 1/22/2013 59 Shigella

Hektoen agar :

Hektoen agar Initially intended for the selective isolation of Shigella spp Can use for Salmonella spp . Contains high levels of Peptone  counteract the toxic effects of the Bile Salts  medium selective 1/22/2013 60 Shigella

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In addition to Lactose, Sucrose and Salicin are also included  improved differentiation A double indicator system of Acid Fuchsin and Bromothymol Blue 1/22/2013 Shigella 61

Salmonella-Shigella (SS) agar :

Salmonella-Shigella (SS) agar 1/22/2013 62 Shigella

SS-Agar:

SS-Agar Meat extract 5.0g Peptone 5.0g Lactose 10g Ox bile, dehydrated 8.5g Sodium citrate 10g Sodium thiosulfate 8.5g Ferric citrate 1g Brillant green 0.0003g Neutral red 0.025g Agar 15g Final pH 7.0 +/- 0.2 at 37°C 1/22/2013 63 Shigella

SS agar:

SS agar Selective medium for Salmonella spp. and Shigella spp.  modification of the original DCA Medium It differs from DCA  extra Bile Salts as well as Sodium Citrate and Brilliant Green dye 1/22/2013 64 Shigella

xylose-lysine-deoxycholate (XLD) agar :

xylose -lysine- deoxycholate (XLD) agar 1/22/2013 65 Shigella

XLD agar contains:

XLD agar contains Yeast extract 3g/l L-Lysine 5g/l Xylose 3.75g/l Lactose 7.5g/l Sucrose 7.5g/l Sodium deoxycholate 1g/l Sodium chloride 5g/l Sodium thiosulfate 6.8g/l Ferric ammonium citrate 0.8 mg/l Phenol red 0.08g/l Agar 12.5g/l 1/22/2013 66 Shigella

MacConkey agar :

MacConkey agar 1/22/2013 67 Shigella

Eosin-methylene blue (EMB) agar:

Eosin-methylene blue (EMB) agar 1/22/2013 68 Shigella

CULTURAL CHARACTERISTICS:

CULTURAL CHARACTERISTICS All members of Shigella are aerobic and facultative anaerobes Grow readily in culture media at pH 6.4 to 7.8 at 10 o C - 40 o C, with optimum of 37 o C After 24 hours incubation, Shigella colonies reaches a diameter of about 2 mm 69 1/22/2013 Shigella

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Colonies circular, convex, colorless, moderately translucent smooth surface, and entire edges Small tangled hair-like projections can sometimes be seen at one or more points on the periphery of the colony 1/22/2013 70 Shigella

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In XLD  pinkish to reddish colonies Hektoen Enteric Agar (HEA),  green to blue green colonies 1/22/2013 Shigella 71

Biochemical Characteristics …..:

Biochemical Characteristics ….. Catalase variable Oxidase negative Indole variable Urease negative Citrate negative 1/22/2013 72 Shigella

Fermentation ….. :

Fermentation ….. Ferment glucose. Lactose non- fermenters except S. sonnei which is a late lactose fermenter Mannitol-fermenters except S . dysenteriae . Produce acid but no gas from fermentable sugars. 1/22/2013 73 Shigella

Biochemical Characteristics …..:

Biochemical Characteristics ….. TSIA – k/a All shigellae H 2 S negative Non motile Nitrate positive Do not form acetyl-methyl carbinol Does not liquefy gelatin 1/22/2013 Shigella 74

Distinguishing features:

Distinguishing features Sub gp A B C D Species Sh. dysenteriae Sh. flexneri Sh. boydii Sh. sonnei Manitol - A A A Lactose - - - A late Sucrose - - - A late Dulcitol - - V - Indole V V V - Ornithine decarboxylase - - - + 1/22/2013 Shigella 75

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Mannitol Indole Serotypes Others S. dysenteriae - - 1,3,4,5,6,10 11,12,13 ONPG + Catalase – Arabinose - S. dysenteriae - + 2,7,8 ONPG + Catalase – Arabinose - S. flexneri (subgroup B) + - 1,2, S. flexneri + + 3,4,5 S. flexneri (subgroup B) + - 6 Manchester & Newcastle Acid + gas +

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Mannitol Indole Serotypes Others S. boydii (subgroup C) + - 1,2,3,4,6,8,10,12,14,18 S. boydii 10, 14 arabinose +, trehalose + 19 is ONPG+ Lactose + S. boydii (subgroup C) + + 5,7,9,11,13,15,16,17 S. sonnei (subgroup D) + _ Lactose + ONPG+ Xylose – Rhamnose +

Other methods….:

Other methods…. PCR ELISA Typing with anti sera Colicin typing  sonnei Phage typing  flexneri Vero and HeLa cell tests 1/22/2013 Shigella 78

PCR :

PCR Sensitive and rapid methodology for identification Shigella species utilizes DNA probes that hybridize with common virulence plasmid genes or DNA primers that amplify plasmid genes by polymerase chain reaction 1/22/2013 Shigella 79

ELISA:

ELISA Using antiserum or monoclonal antibody  Ipa proteins to screen stools Technique useful for epidemiological studies Too specialized for routine use 1/22/2013 Shigella 80

Serotyping :

Serotyping Should be performed from a non–sugar-containing medium, such as 5% sheep blood agar Preliminary serologic grouping of Shigella spp. Is also performed using commercially available polyvalent somatic (“O”) antisera designated A, B, C, and D Subtyping of Shigella spp. beyond the groups A, B, and C performed by reference laboratories 1/22/2013 81 Shigella

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Non lactose/ late lactose fermenting Non motile organisms Mannitol Negative Mannitol positive Shigella dysenteria Non lactose fermenter Late lactose fermenter Indole positive Indole negative Shigella boydii Shigella flexneri Shigella sonnei 1/22/2013 82 Shigella LAB DIAGNOSIS

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1/22/2013 Shigella 83 Mannitol Negative Mannitol Negative Late lactose fermenter Non lactose fermenter S.dysenteriae polyvalent Positive Positive Positive Negative Negative Negative S. Flexnrri polyvalent Negative Reference lab Discard Discard Discard S. Sonnei S. Boydii polyvalent Indole negative Indole positive

Viability :

Viability Cultures retain viability  many years  Dorset egg Killed by moist heat at 55 0 C in 1 hr Killed by 1% phenol in 15 mts Die within few hrs in feaces Can survive in buffered glycerol solution / at 4 0 C 1/22/2013 Shigella 84

TREATMENT:

TREATMENT Shigella dysentery  Sh. Sonnei  do not require antibiotic therapy Maintenance of good nutrition Maintenance of hydration by use of oral rehydration salt solution Treatment with a suitable antibiotic  very young, the aged or the debilitated 1/22/2013 85 Shigella

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Treatment with ciprofloxacin is indicated in severe cases Ampicillin , tetracyclines and trimethoprim are suitable alternatives Resistance common 1/22/2013 Shigella 86

ORS:

ORS Constituent Amount (g ) Sodium chloride 2.6 Potassium chloride 1.5 Trisodium citrate 2.9 Glucose (anhydrous) 13.5 1/22/2013 87 Shigella

Shigellosis :

Shigellosis Shigellosis a major problem in tropical areas of the developing world  shigellosis is endemic 5 million cases require hospital treatment and 600000 die every year Young children are particularly vulnerable 1/22/2013 88 Shigella

Sources and spread:

Sources and spread Highly contagious and is usually spread by the faecal-oral route Contamination of foods  those consumed raw 1/22/2013 89 Shigella

EPIDEMIOLOGY:

EPIDEMIOLOGY During the twentieth century, infections due to S. dysenteriae 1 and S. boydii declined and strains of S. sonnei became dominant in the UK and other European countries In England and Wales more than 16000 cases of Sonnei dysentery were recorded in 1992. 1/22/2013 90 Shigella

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The incidence then declined steadily to an annual average of fewer than 1000 notified cases between 1998 and 2004 1/22/2013 Shigella 91

Distribution of serogroups and serotypes :

Distribution of serogroups and serotypes S. flexneri and S. Sonnei endemic form of shigellosis S. dysenteriae 1 deadly epidemics S. boydii is rarely found developing areas  S . Flexneri serotypes 1b, 2a, 3a, 4a, and 6. 1/22/2013 92 Shigella

Prevention of shigellosis :

Prevention of shigellosis Interruption of disease transmission Avoiding contact with contaminated faeces Clean water and good sanitary conditions 1/22/2013 93 Shigella

Prevention of shigellosis.....:

Prevention of shigellosis..... Safe disposal of excreta, food protection, and person-to-person protection by hand washing, and elimination/protection against flies 1/22/2013 Shigella 94

Prevention of shigellosis.....:

Prevention of shigellosis..... Promote the use of oral rehydration therapy to offset the effects of acute diarrhoea 1/22/2013 Shigella 95

Current Research :

Current Research Vaccine development: Human monocyte -derived dendritic cells (DC) Vaccine under study Mice used as models Incubation of DC with inactivated Shigella clearly induced immune reactions Mice immunized orally or intranasally with inactivated S. flexneri S. sonnei , or S. dysenteriae 1/22/2013 96 Shigella

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When later exposed to LPS Shigella bacterial cell  developments of IgG and IgA When infected with live homologous organisms  80-100% survival in all vaccinated groups Negligible survival in control groups 1/22/2013 97 Shigella

IMPORTANT EPIDEMICS :

IMPORTANT EPIDEMICS 1/22/2013 Shigella 98

Shigellosis :

Shigellosis 14 July 2004 5 November 2003 5 November 2003 4 February 2000 28 January 2000 24 January 2000 Shigellosis in Sudan Shigellosis in Central African Republic Bloody diarrhoea in Liberia Dysentery in Sierra Leone Dysentery in Lesotho Dysentery in Sierra Leone 1/22/2013 99 Shigella

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1/22/2013 Shigella 100 Tha nks

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