Microbiological aspects of Salmonella including morphology, culture characters, virulence factors, carrier state and prevention.

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2 Dr. Waleed Eldars

3 Microbiological aspects of Salmonella including morphology, culture characters, virulence factors, carrier state and prevention. Microbiological aspects of Brucella including morphology, culture characters, and prevention.

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5 Gram-negative facultative anaerobe non lactose fermenter bacilli. Salmonellae live in the intestinal tracts of humans and animals.

6 Serological characters

7 -O (Somatic or cell wall antigen). -H (flagellar antigen). -Vi (polysaccharide virulence).

8 Somatic (O) or cell wall antigens, are heat stable and alcohol resistant. Surface antigens: may be found in some Salmonella serovars. One specific surface antigen is the Vi antigen which occurs in S typhi and Paratyphi c. Flagellar (H) antigens: Flagellar antigens are heat-labile proteins.

9 Pathogenicity 1- Enteric fever (Typhoid fever, Paratyphoid fever) (S. typhi, S. paratyphi A,B,C). No animal reservoir. 2- Food poisoning (Gastroenteritis) S. typhimurium, S. enteretidis). No systemic infection. Zoonotic infection. 3- Septicemia S. cholerasuis.

10 may be caused by any species but S. cholerae-suis is common. In this case, antibiotic therapy is required

11 It is the most common salmonella infection. Caused by S. typhimurium and S. enteritidis. The organisms invade the epithelium and do not produce systemic infection.

12 There is no human reservoir, animal reservoir is very important. Transmitted from contaminated food (poultry, meat, dairy products). Incubation period is hours after ingestion of contaminated food.

13 Symptoms are diarrhea, vomiting and fever that last 2-5 days. Prevention by Avoiding contamination of foods, Improvement of hygiene, Use of pasteurized milk and milk products Proper storage of foods.

14 Typhoid fever is caused by S.typhi & is strictly a human disease. Paratyphoid fever is caused by S.paratyphi A or B or C. Transmitted from human reservoir by contaminated water or food. Source is case or carrier. A carrier state is common; thus a food handler can cause a lot of spread.

15 The bacteria enter human digestive tract, penetrate intestinal mucosa and multiply in mesenteric lymph nodes, passes into blood Bacteriaemia usually within the first week. The Bacteriaemia is temporary and the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks.

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17 Release of endotoxin and exotoxins. Salmonella strains may produce a thermolabile enterotoxin. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid.

18 Salmonella excretion by human patients may continue long after clinical cure. Asymptomatic carriers are potentially dangerous. About 5% of patients clinically cured from typhoid remain carriers for months or even years. Antibiotics are usually ineffective on Salmonella carriage because the site of carriage in gall bladder may not allow penetration by the antibiotic.

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20 Laboratory Diagnosis of Enteric Fever

21 I. Diagnosis of A case of Enteric Fever

22 A)Isolation of the organism: First Weak Second Weak Third Weak Blood Stool Stool Urine

23 2. Culture of faeces: In at least 50% of the cases culture of faeces is positive in first week. The isolation of S. typhi greatly increases in the second or third week.

24 3. Urine culture: Urine culture is positive in one third of cases, and in great majority of cases, it will be free from organisms weeks before the faeces negative. becomes

25 Cultural characters O2 facultative anaerobe Temp 37 C Culture characters CO2

26 Enrichment Selenite broth tetrathionate Nutrient agar Media Selective SS, HE, XLD Indicator Mackonkey, DCA

27 On MacConkey and DCA media: they produce pale colonies (NLF). Maximal recovery of salmonella is obtained by using enrichment broth medium as selenite or tetrathionate broth.

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29 1. Film stained by Gram: Gram-negative bacilli. motile. non capsulated. non sporulated.

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31 2. Biochemical reactions: Ferment glucose, mannitol, maltose, and sorbitol with acid (S. typhi) and gas (S. paratyphi). Lactose is not fermented. Negative Indole, Voges-Prosk and Urease tests. H2S produced from thiosulfate.

32 By using O and H antisera against salmonellae to confirm the diagnosis.

33 b)serodiagnosis: Widal test ( tube agglutination test): Use: Widal test is an agglutination test for detection of antibodies against Salmonella typhi and Salmonella paratyphi, the common causal agents of enteric fevers. Diagnositic titer: 80.

34 When serum sample containing antibodies against S.typhi and S.paratyphi A, B or C are mixed with respective antigens, agglutination will take place. In S.typhi and S.paratyphi two types of antigens are recognized as diagnostically important: O antigen Somatic antigen. H antigen Flagellar antigen.

35 O antigens of various species have components in common and hence only one O antigen i.e. non species specific. H antigens of Salmonella spp. are species specific, and hence the H antigens of all S.typhi, S.paratyphi A and S.paratyphi B and S.paratyphi C are employed in the test. Serum antibodies against H and O antigens of salmonella usually appear by the 7th-10th day of infection and the titer reaches maximum during the 4th week.

36 O Ab H Ab typhi H Ab paratyphi A H Ab paratyphi B H Ab paraty phi C Result S. Typhi inf S. P.typ. A inf S. P.typ. B inf Recent vaccine Old vaccine

37 II. Diagnosis of Typhoid Carrier

38 - 5% of cured - Asymptomatic transmission. - Difficult treatment. In order to label a person as a typhoid carrier, the isolation of the organism should be done from urine or faeces. If these are repeatedly negative, bile or duodenal aspirate can be used. Vi antibodies present in a titre of more than 1:10 is also suggestive of chronic typhoid carrier.

39 Vaccination against typhoid fever

40 1.Live oral vaccines: Using living avirulent bacteria. It is given in four doses, 2 days apart, as needed for protection. A booster dose is needed every 5 years for people who remain at risk. Should not be given to children younger than 6 years of age.

41 2.The parenteral heatphenolinactivated vaccine (TAB): Has been widely used for many years. Given in two doses. Efficacy over 2-3 year after vaccination ranges from 50% to 77%. A booster dose is needed every 2 years for people who remain at risk.

42 3. The newly licensed parenteral vaccine [Vi capsular polysaccharide (ViCPS) Composed of purified Vi ( virulence ) antigen, elaborated by S.Typhi

43 4. An acetoneinactivated parenteral vaccine: Currently available only to the armed forces in America. Efficacy for this vaccine, ranges from 75% to 94%. NOTE: No typhoid vaccine is 100% effective and is not a substitute for being careful about what you eat or drink.

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45 Three species are important human pathogens: B. abortus affects cows. B. melitensis affects goats and sheep. B. suis affects pigs. Species are differentiated by production of urease and H 2 S, dye sensitivity, cell wall antigens and phage sensitivity.

46 Gram-negative cocco bacilli Non-spore-forming Non-motile Non capsulated.

47 Brucellosis is a severe acute febrile disease caused by Brucella species. Human infections are acquired from handling of infected animals Consuming contaminated milk or milk products (zoonosis ( No human to human transmission.

48 Acquired Exposure is frequently occupational. Thus, veterinarians, meat workers and animal handlers are at great risk. In animals, brucellae affect the reproductive organs causing abortion and sterility. In contrast to animals, abortion is not a feature of brucellosis in pregnant women.

49 Portals of entry are the mouth, conjunctivae, respiratory tract and skin abrations. Brucellae are facultative intracellular parasites, multiply in monocyte-macrophage cells to reticuloendothelial system (spleen, liver, bone marrow, lymph nodes and kidneys), where they live and multiply forming granulomas in these organs.

50 Release of brucella from granulomas causes recurrent bacteraemia and recurrence of fever and chills. These may produce an undulant fever in which intensity of fever and symptoms recur and recede at about 10 day intervals.

51 Diagnosis is based on: prolonged presence of undulating fever (at least a week) history of exposure

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53 Specimens: Blood, biopsy of (lymph node, spleen, liver and bone marrow), samples are highly infectious (hazard group 3) should be handled in a safety cabinet.

54 Blood culture : the method of choice early in the disease.

55 O2 Strict aerobes Duration 3-4 w Culture characters CO2 5-10% B. abortus Temp 37 C

56 Media: enriched medium is needed to support adequate Brucella growth. Tryptone soya castenada diphasic medium are used for brucellae isolation from blood

57 Morphology Molecular Identification of growth: BR Slide aggl

58 Morphology: G-ve, nonmotile, coccobacilli. Biochemical reaction: Catalase +ve oxidase +ve H2Sproduction +ve for B. abortus & B. suis. Slide agglutination with specific antiserum and growth inhibition by dyes Molecular techniques for typing are being developed.

59 Organism Br melitensis Inhibition of growth by Basic Fuchsin 1/25000 Thionine 1/30000 CO2 required H2S production Br abortus Br suis + +

60 1. Rapid slide agglutination Rapid screening. Undiluted and diluted serum used to avoid prozone phenomenon

61 2. Tube agglutination test: Wide range of dilution of the patient serum (up to 1/1280) to avoid prozone phenomenon, A titer of 1/100 to 1/200 is diagnostic, interpretation depends on patient occupation and disease endemicity, Rising titer is diagnostic.

62 Agglutination

63 Prozone phenomenon A falsely negative test due to very high titers (concentration) of antibody.

64 3. EIA (enzyme immunoassay) test: detects specific IgM and IgG

65 C)PCR: direct detection of brucella in clinical samples D)Brucellin test: Principle: intradermal allergic test similar to tuberculin Method: using heat killed brucella suspension for intradermal injection

66 Pasteurizing milk Minimise occupational exposure by observing safety precautions (protective clothing and laboratory containment). Eradication of infected animals and vaccination of animals by live attenuated vaccines to reduces the reservoir. Vaccines for humans have been developed. Vaccination for persons at high risk is possible, but not widely accepted. 3/19/

67 Tetracycline. OR: A tetracycline/streptomycin combination is generally curative. 3/19/

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