1. بسم الله الرحمن الرحيم

2. Assistant professor of microbiology & immunology
Wafaa khalil zaki
Assistant professor of microbiology & immunology

3. Yersinia & Pasteurella

4. Intended learning outcomes
By the end of the lecture students should be able to:
1- Recognize morphology ,cultural characteristics of the Yersinia & Pasteurella
group
2- Understand pathogenesis and virulence factors of pathogenic members and their epidemiology
3-Demonstrate clinical types, microbiological diagnosis prevention and treatment of them

5. These organisms are short Gram negative coccobacilli which exhibit bipolar staining with certain stains, methylene blue & Leishman stain. Animals are their natural hosts but they can produce severe diseases in humans.

7. Classification: Species of medical importance are: Yersinia pestis.
Yersinia pseudotuberculosis & Yersinia enterocolitica.
Pasteurella multocida.

8. Yersinia pestis and Plague
Yersinia pestis (Y. pestis) causes a serious disease called plague (Black Death).
Pathogenesis and Virulence Factors:
Virulence factors of Y. pestis include:
Polypeptide capsule that is formed at 37˚C, it is anti-phagocytic and anti- complementary.

9. Endotoxin
Plasminogen activator promotes the dissemination of the organism. It also destroys C3b on the bacterial surface, thus attenuating phagocytosis.

11. Plague is primarily a disease of rodents
Plague is primarily a disease of rodents. A flea feeds on an infected rodent, the ingested organisms loses its capsule, multiply in flea gut & produces coagulase enzyme at 27˚C that blocks the flea proventriculus.

13. The "blocked and hungry flea bites repeatedly other hosts including human and regurgitates the infected material into the bite wound

15. The bulk of non-capsular organisms are phagocytized and destroyed by neutrophils. However, few organisms are taken up by macrophages and histiocytes which are unable to kill them and allow them at 37˚C to re synthesize their capsule and multiply. when they are released from histiocytes they are resistant to phagocytosis and killing by neutrophils.

16. After several days an acute inflammatory reaction develops in the draining lymph nodes that become markedly enlarged and tender (buboes). The infection spreads through the lymph to the thoracic duct and allowing large numbers of virulent organisms to reach the blood stream. This stage is called septicemic plague.

18. Endotoxin release results in shock and disseminated intravascular coagulation. From blood, the bacilli reach many organs especially the lungs causing secondary pneumonic plague.

20. The fully virulent organisms can spread from a case with pneumonic plague to another susceptible human (primary pneumonic) which is highly fatal.

21. Epidemiology Plague is enzootic in wild rodents; transmitted by fleas.
Epizootics occur intermittently; at such times, the infection can spread to domestic rodents (e.g. rats) and is transmitted among them by rat fleas (Xenopsylla cheopis) (urban plague)..

22. Infection is transmitted from infected rats to humans by flea bites mainly. In case of pneumonic plague spread can occur from human to human by respiratory droplets (epidemic plague)

23. Clinical Types: There are three main clinical forms of the disease:
Bubonic plague:
The organism multiplies in the draining lymph nodes. This is the commonest form and is characterized by high fever and acute lymphadenitis with painful haemorrhagic lymph nodes called buboes.

24. Pneumonic plague:
1ry pneumonic plague: Severe haemorrhagic bronchopneumonia after inhalation of the organism. It can be spread by aerosols in bioterrorism.
2 ry pneumonic plague : spread to the lungs via the blood stream.
It is highly infectious and sputum is often blood stained & contains large numbers of Y. pestis bacilli.

25. Septicemic plague:
A serious haemorrhagic fatal condition in which large number of Y. pestis bacilli are present in the blood. The organism causes diffuse intra vascular coagulation resulting in intra vascular thrombi and purpuric lesion.

26. The disease is known in the middle ages as the black death
The disease is known in the middle ages as the black death. This is because it frequently leads to gangrene and blackening of various parts of the body. Capillary fragility results in hemorrhages in the skin which also result in black patches.

27. Laboratory Diagnosis of Plague:
specimen >>>>>BSL 2/3
Blood for blood culture in septicemic plague.
Aspirate from lymph node (buboes) in bubonic plague.
Bronchial washings in pneumonic plague.
Microscopy

29. Direct & rapid detection
Detection of capsular antigen
(Mainly on supernatant of CSF)
Direct or indirect immuno- fluorescence
PCR

30. Culture:
The organism can grow on sheep blood agar or sulphite agar medium forming small shiny non hemolytic colonies after 24 – 48 hours incubation at 27oC which is the optimum temperature for growth. It gives non-lactose fermenting colonies on

31. MacConkey’s agar medium (incubated anaerobically) which disappear after 2-3 days as a result of autolysis.

33. Colonies are further identified by:
Morphology based on Gram and Leishman`s stain stained films
Biochemical reactions
Definite identification is best made by immunofluorescence
Animal inoculation: exudate from bubo or 24hr broth culture is injected S.C into laboratory animals as guinea pigs, white rat or mice.

34. Animals die within a few days with septicaemic plague (highly pathogenic to laboratory animals).
Leishman stained films made from spleen, liver or heart blood show numerous plague bacilli.

35. Serological Diagnosis:
Used in case of negative cultures. Detection of a rising titer in two sequential specimens confirms the diagnosis.
Rapid methods:
are the methods of choice when available for safety and rapid diagnosis.
ELISA &PCR (detect F1 capsular antigen)

36. Treatment:
IM Streptomycin is the drug of choice. Tetracycline is an alternative drug
Sometimes the two drugs are given in combination

37. Prevention and control
Eradication of plague infected rodents.
Anti-rat measures including fumigation of ships & aircrafts
Anti-flea measures (insecticides).
Chemoprophylaxis for contacts of cases with pneumonic plague (e.g., Tetracyclines).
Formalin killed vaccine for persons at risk

38. What are the pathogenesis and clinical forms of plague?

39. Yersinia enterocolitica & Yersinia pseudotuberculosis

40. Epidemiology:
These organisms are found in the intestinal tract of a variety of animals (domestic and farm animals). Human infection results from ingestion of food contaminated with animal feces.

41. Pathogenesis:
During the incubation period of 5-10 days, the organisms multiply in the gut mucosa particularly the ileum leading to inflammation and ulceration. The process may extend to mesenteric lymph nodes

42. Clinical Findings:
Infection results in enterocolitis (yersinosis) and mesenteric lymphadenitis, characterized by fever, abdominal pain, and diarrhea which ranges from watery to bloody. Sometimes the patients present with severe pain in the right iliac fossa simulating appendicitis.

43. Post infectious immune disease: maybe complicated after 2 weeks by reactive arthritis or erythema nodosum.

44. Laboratory Diagnosis
Specimen: blood, stool or material obtained at surgical exploration.
Direct smear stained with:
Gram stain: shows small gram negative bacilli.
Leishman's stain: shows bipolar stained bacilli (safety pin appearance) .

45. Blood agar: non haemolytic colonies.
Motility test: motile at 25 oC but not at 37 oC.
Culture at 25 oC on:
Blood agar: non haemolytic colonies.
MacConkey's agar: non lactose fermenting colonies.
Biochemical reactions: oxidase negative and urease positive
ELISA detects antibodies

47. Treatment :
The disease is usually self-limited. Tetracyclines or aminoglycosides are used in severe cases.

48. Pasteurella multocida
This organism occurs worldwide in the respiratory and GIT of many domestic and wild animals. It causes in humans "animal bite fever", a local abscess at the site of bite wounds from animals especially cats and dogs.

49. Diagnosis:
Culture on blood agar at 37C. Identified by Gram stained film which shows capsulated Gram negative bacilli with bipolar staining and biochemical tests.
Treatment:
Penicillin G is the drug of choice.
Tetracycline and fluoroquinolones can be used as alternatives

50. Francisella tularensis
Francisella tularensis is the causative organism of tularemia (rabbit fever). It is widely distributed in animal reservoirs. It is a typical zoonosis

51. Mode of Transmission: Biting arthropods e.g. ticks and deer fleas.
Direct or indirect contact with infected animal tissues
Inhalation of aerosols (biowarfare or laboratory acquired)
Ingestion of contaminated food or water.

53. Morphology:
The organisms are capsulated Gram negative pleomorphic rods with bipolar staining. They are strict aerobes. They do not grow on ordinary bacteriological media.

55. Pathogenesis:
Francisella tularensis is highly infectious. Penetration of skin or mucus membranes or inhalation of organisms can result in infection. It is an intracellular organism that multiplies within phagocytic cells and spreads to different parts of the body via lymphatics and blood vessels.

56. Dissemination of the organisms through the bloodstream permits focal lesions to develop in numerous organs. The patient will normally exhibit one of several clinical syndromes depending on route of transmission.

57. Clinical Findings:
Ulcerogalndular form: It is the most common ( %) in which a painful ulcerating papule which has a necrotic center and raised periphery develops at the site of infection.
Glandular form: Lymphadenopathy without ulcer
Oculoglandular form.

58. Pneumonic form: Influenza-like illness (acute onset of fever, headache, rigors & painful pharyngitis that progress to pneumonia with high mortality rate. Pneumonia occurs when the organisms infect the lungs from the blood stream or by inhalation.
Typhoidal form: typhoid-like illness

59. Laboratory diagnosis
Specimen: Sputum, ulcer swab, lymph node aspirate.
Culture requires BSL-2/3.
Specimen is cultured on glucose cysteine blood agar aerobically at 37°C. The organism is identified by immunofluorescence or agglutination by specific antisera.

60. ELISA : the diagnosis rests on serological In generstudies through a rise in agglutination titer in paired serum samples collected two weeks apart.
A single titer of 1/160 is highly suggestive if history and physical findings are compatible with the diagnosis.
PCR

61. PREVENTION
Taking the appropriate biosafety precautions when working with the organism in laboratory.
A live attenuated vaccine is available for laboratory workers and others at high risk for infection (occupational).

62. Treatment:
Streptomycin or Gentamicin for 10 days is usually effective.
Prevention and Control:
Insect repellants and protective clothing (esp. when in woods, forests etc) to avoid insect bites
Tick control.

63. Thank you