1. Bartonella Prepared by: Ohood R. Sarsour Ohood R. Sarsour

2. Introduction Bartonella species (formerly known as Rochalimaea) Bartonella species (formerly known as Rochalimaea) Linked to a number of emerging zoonotic diseases Linked to a number of emerging zoonotic diseases B.quintana, B.bacilliformis, B.henselae B.quintana, B.bacilliformis, B.henselae Unique and common feature is ability to cause vasoproliferative lesions (process of pathological angiogenesis) Unique and common feature is ability to cause vasoproliferative lesions (process of pathological angiogenesis) Infection affects function of endothelial cells resulting in cell invasion, suppression of apoptosis and induction of proliferation, resulting in vasoproliferative tumor growth Infection affects function of endothelial cells resulting in cell invasion, suppression of apoptosis and induction of proliferation, resulting in vasoproliferative tumor growth

3. B. henselae Gram negative aerobic rods Gram negative aerobic rods Main reservoir is cats Main reservoir is cats Transferred to humans through scratches and bites (fleas/ticks??) Transferred to humans through scratches and bites (fleas/ticks??) Resulting illness is Cat Scratch Disease (CSD) Resulting illness is Cat Scratch Disease (CSD) About 40% of cats carry B.henselae About 40% of cats carry B.henselae Cats are asymptomatic, although bacteria can be cultivated from their blood for up to 2 years Cats are asymptomatic, although bacteria can be cultivated from their blood for up to 2 years This persistent infection due to persistence in red blood cells This persistent infection due to persistence in red blood cells

4. Epidemiology Cases of CSD occur worldwide Cases of CSD occur worldwide Higher incidence of Bartenellosis in warmer and more humid areas of the United States Higher incidence of Bartenellosis in warmer and more humid areas of the United States More fleas and ticks More fleas and ticks Children 2-24 and veterinarians make up 80-90% of human cases Children 2-24 and veterinarians make up 80-90% of human cases 22 000 cases of CSD are diagnosed per year in the United States 22 000 cases of CSD are diagnosed per year in the United States Most cases occur in late fall and winter Most cases occur in late fall and winter

5. Infections CSD is a self-limiting disease characterized by lymphadenopathy and infection at the site of injury CSD is a self-limiting disease characterized by lymphadenopathy and infection at the site of injury Lasts 6-12 weeks in healthy individuals and can be accompanied by headaches, nausea, vomiting and fatigue Lasts 6-12 weeks in healthy individuals and can be accompanied by headaches, nausea, vomiting and fatigue Severity of CSD depends on immune status of the host Severity of CSD depends on immune status of the host Immunocompromised individuals can develop Bacillary angiomatosis (BA) and Parinaud’s oculoglandular syndrome (POS) Immunocompromised individuals can develop Bacillary angiomatosis (BA) and Parinaud’s oculoglandular syndrome (POS)

6. Diagnosis is difficult and is usually based on exposure to a cat, enlarged lymph nodes and lack of another appropriate cause Diagnosis is difficult and is usually based on exposure to a cat, enlarged lymph nodes and lack of another appropriate cause

7. Virulence Factors VEGF (vascular endothelial growth factor) VEGF (vascular endothelial growth factor) Triggers angiomatosis and has a pathogenic role in vasoproliferative disorders (such as BA) Triggers angiomatosis and has a pathogenic role in vasoproliferative disorders (such as BA) Highly mitogenic to endothelial cells Highly mitogenic to endothelial cells B.henselae induces host cell production of VEGF B.henselae induces host cell production of VEGF This production and subsequent proliferation is necessary for angiogenesis and BA This production and subsequent proliferation is necessary for angiogenesis and BA This proliferation also promotes growth of B.henselae’s own cells This proliferation also promotes growth of B.henselae’s own cells

8. Pili Pili Expression of pili allows adhesion and invasion of host cells Expression of pili allows adhesion and invasion of host cells Pili are also thought to be responsible for the induction of VEGF as pili - mutants have a significant decrease in production of VEGF Pili are also thought to be responsible for the induction of VEGF as pili - mutants have a significant decrease in production of VEGF

9. Type 4 secretion system (virB-D4) Type 4 secretion system (virB-D4) Consists of a multiprotein channel that allows transportation of DNA or protein into the host cell Consists of a multiprotein channel that allows transportation of DNA or protein into the host cell virB operon encodes 10 genes, 8 of which known to encode a T4SS virB operon encodes 10 genes, 8 of which known to encode a T4SS virD4 locus located just downstream virD4 locus located just downstream virB5 encodes a 17kD protein found in patients with CSD virB5 encodes a 17kD protein found in patients with CSD Function not known but not found outside Bartonella species Function not known but not found outside Bartonella species

10. Downstream region of virB encodes Bep proteins that are translocated into the endothelial cells Downstream region of virB encodes Bep proteins that are translocated into the endothelial cells These proteins thought to mediate all virB-virD4- dependent changes of endothelial cell function These proteins thought to mediate all virB-virD4- dependent changes of endothelial cell function Recent studies have shown that a mutation in either virB or virD4 prevents intraerythrocytic infection as well as endothelial cell invasion Recent studies have shown that a mutation in either virB or virD4 prevents intraerythrocytic infection as well as endothelial cell invasion

11. Type 4 Secretion System (Trw) Type 4 Secretion System (Trw) Second T4SS identified that is active during endothelial cell infection Second T4SS identified that is active during endothelial cell infection Primary function to establish contact with erythrocytes through surface expressed T4SS pili Primary function to establish contact with erythrocytes through surface expressed T4SS pili

12. Deformin Deformin Hydrophobic secreted protein Hydrophobic secreted protein Affinity for albumin Affinity for albumin Formation of pits and trenches on red blood cell membranes that aids in colonization and entry to the cell Formation of pits and trenches on red blood cell membranes that aids in colonization and entry to the cell

13. Outer Membrane Proteins (OMPs) Outer Membrane Proteins (OMPs) Induce NF κ B-dependent upregulation of E- selectin and ICAM-1 Induce NF κ B-dependent upregulation of E- selectin and ICAM-1 Leads to increased adhesion molecule expression Leads to increased adhesion molecule expression NF κ B plays a role in initiation and regulation of the body’s proinflammatory response NF κ B plays a role in initiation and regulation of the body’s proinflammatory response

14. Persistent Infection Endothelial cell interaction Endothelial cell interaction Primary niche Primary niche Most information gathered using HUVEC cells Most information gathered using HUVEC cells Bartonella species can enter endothelial cells by rearranging actin cytoskeleton Bartonella species can enter endothelial cells by rearranging actin cytoskeleton B.henselae can also enter using “invasome” mechanism B.henselae can also enter using “invasome” mechanism Bacterial aggregate formation on cell surface that is engulfed and internalized through an actin- dependent process Bacterial aggregate formation on cell surface that is engulfed and internalized through an actin- dependent process

16. Intraerythrocytic persistence Intraerythrocytic persistence Common theme of Bartonella is prolonged intraerythrocytic bacteremia associated with transmission by blood-sucking arthropods Common theme of Bartonella is prolonged intraerythrocytic bacteremia associated with transmission by blood-sucking arthropods Adherence mediated by pili and OMPs exposed on bacterial surface Adherence mediated by pili and OMPs exposed on bacterial surface Exact mechanism is not known but assumed to be bacterial-induced endocytosis Exact mechanism is not known but assumed to be bacterial-induced endocytosis Assisted by deformin secretion Assisted by deformin secretion

17. After invasion intracellular multiplication occurs After invasion intracellular multiplication occurs Limited by quorum sensing to prevent hemolysis Limited by quorum sensing to prevent hemolysis Bacteria survive for the duration of the cell life and remain in the blood stream for several weeks, facilitating transmission Bacteria survive for the duration of the cell life and remain in the blood stream for several weeks, facilitating transmission Bacteria seeded into blood stream every 5 days Bacteria seeded into blood stream every 5 days Unique persistence is an adaptation to mode of transmission Unique persistence is an adaptation to mode of transmission Intraerythrocytic bacteremia subsides due to strong IgG antibody response (in animal models) Intraerythrocytic bacteremia subsides due to strong IgG antibody response (in animal models)

19. Prevention and Treatment Proper cat care Proper cat care Keeping cats indoors Keeping cats indoors Using flea products Using flea products Prevent roughhousing with kittens Prevent roughhousing with kittens Do not let cats like open wounds Do not let cats like open wounds Wash cat wounds out with warm water Wash cat wounds out with warm water

20. Most healthy people recover without treatment in anywhere from 3 weeks to several months Most healthy people recover without treatment in anywhere from 3 weeks to several months Immunocompromised individuals may develop more serious complications and possibly death Immunocompromised individuals may develop more serious complications and possibly death If antibiotics are prescribed If antibiotics are prescribed Erythromycin, rifampin, doxycycline Erythromycin, rifampin, doxycycline

21. Antibiotics can not reach intraerythrocytic bacteria but can prevent new waves of parasitism Antibiotics can not reach intraerythrocytic bacteria but can prevent new waves of parasitism A recent study has shown that cats treated for bacteremia caused by B.henselae were resistant to reinfection when challenged following recovery A recent study has shown that cats treated for bacteremia caused by B.henselae were resistant to reinfection when challenged following recovery

22. B.quintana Facultative,intracellular,gram negative rod. Facultative,intracellular,gram negative rod. Catalase and oxidase reactions are negative. Catalase and oxidase reactions are negative. The bacterium can be grown on axenic media The bacterium can be grown on axenic media When grown on blood agar,rough colonies embedded in the agar obtained after 12to14 days When grown on blood agar,rough colonies embedded in the agar obtained after 12to14 days Humans are the reservoir of the bacterium,and the human body louse is its usual vector Humans are the reservoir of the bacterium,and the human body louse is its usual vector The bacterium has atropism for endothelial cells,leading to angioproliferative lesions,observed in bacillary angiomatosis. The bacterium has atropism for endothelial cells,leading to angioproliferative lesions,observed in bacillary angiomatosis.

23. Transmission B.quintana is transmitted by the human body louse, which lives in clothes B.quintana is transmitted by the human body louse, which lives in clothes

24. Clinical Manifestations Trench Fever. Trench Fever. Chronic Bacteremia. Chronic Bacteremia. Endocarditis. Endocarditis. Bacillary Angiomatosis. Bacillary Angiomatosis. Lymphadenopathy. Lymphadenopathy.

25. Diagnosis Serologic tests Serologic tests Western blot and cross-adsorption results in a patient with Bartonella quintana endocarditis. A) Nonadsorbed. B) Adsorbed with B. quintana. C) Adsorbed with B. henselae. Lane 1, B. quintana; lane 2, B. henselae; lane 3, B. elizabethae; lane 4, B. vinsonii subsp. Berkhoffi; lane 5, B. vinsonii subsp. Arupensis. Before adsorption (A), antibodies are detected against all species (1, 2, 3, 4, and 5). After adsorption with B. quintana antigen (B), all antibodies disappear. After adsorption with B. henselae antigen (C), antibodies against B. quintana (1) persist. This reaction shows B. quintana infection.

26. Culture. Culture. Molecular Biology. Molecular Biology. Immunohistochmistry and Immunoflurescence. Immunohistochmistry and Immunoflurescence.. Immunohistochemical demonstration of Bartonella sp Laser confocal microscopy showing the intraerythrocytic location of Bartonella quintana.

27. Antimicrobial drug susceptibility of B. quintana Evaluation of susceptibilities to antimicrobial drugs has been performed in both axenic media and cell culture. Evaluation of susceptibilities to antimicrobial drugs has been performed in both axenic media and cell culture. Penicillines,cephalosporins,aminoglycosides,chlo ramphenicol,tetracyclines,rifampin,fluoroquinolo nes,and cortimonazole. Penicillines,cephalosporins,aminoglycosides,chlo ramphenicol,tetracyclines,rifampin,fluoroquinolo nes,and cortimonazole. However only aminoglycosides have bactericidal effect. However only aminoglycosides have bactericidal effect.

28. Bartonella bacilliformis Gram negative aerobic, pleomorphic, flagellated, motile, coccobacillary, 2-3  m large and 0,2 - 0,5  m wide and facultative intracellular bacterium. For its isolation, special cultures are required containing complemental soy agar, proteases, peptones, some essential amino acids and blood. The optimum growing temperature is 19-29 ºC.

29. Suspected vectors: Phlebotomine sand flies Lutzomyia verrucarum Smaller than a mosquito, larger than a midge Coloration varies from light brown (sandy or fawn) to gray or black Require humid, not wet, conditions Only female sand flies take a blood meal Nocturnal feeding behavior

30. Suspected Vectors: Phlebotomine sand flies Lutzomyia peruensis Sand fies are weak fliers Fly only at night unless disturbed in their daytime resting site Sand flies transmit Bartonella bacilliformis from infected to uninfected hosts by bite At least two species suspected in Peru: Lu. verrucarum and Lu. peruensis

31. Pathogenesis Bartonella bacilliformis is transmitted by the bite of the suspected vector Lutzomyia spp Bartonella bacilliformis is transmitted by the bite of the suspected vector Lutzomyia spp Following transmission, the bacterium infects red blood cells and endothelial cells Following transmission, the bacterium infects red blood cells and endothelial cells The physical damage and introduction of antigens in the membranes of the red cells stimulate the Reticuloendothelial System to produce an intense erythrophagocytosis by macrophages and histiocytic cells resulting in severe extra vascular hemolytic anemia The physical damage and introduction of antigens in the membranes of the red cells stimulate the Reticuloendothelial System to produce an intense erythrophagocytosis by macrophages and histiocytic cells resulting in severe extra vascular hemolytic anemia

32. The disease The clinical symptoms of bartonellosis are pleomorphic and some patients may be asymptomatic The clinical symptoms of bartonellosis are pleomorphic and some patients may be asymptomatic The two classical clinical presentations are the acute phase and the chronic phase, corresponding to the two different host cell types invaded by the bacterium The two classical clinical presentations are the acute phase and the chronic phase, corresponding to the two different host cell types invaded by the bacterium

33. Acute phase: Oroya fever or Carrion’s disease The mean incubation time is 21 days (range 10 to 270 days) The mean incubation time is 21 days (range 10 to 270 days) The diagnostic tests in this phase are: The diagnostic tests in this phase are: Values in porcentaje

34. The diagnosis The diagnosis in the acute phase can be done using the thin blood film with Giemsa stain. It is possible to observe the bacillus inside the red blood cells.

35. Lane A: Positive control pool Lane Band C: Bartonella bacilliformis- positive serum taken from a patient in acute phase Lane D: Negative control pool Immunologic technics: Sonicated immunoblot A B C D 20 kDa 18 kDa 17 kDa 14 kDa

36. M: DNA ladder (100 bp). 1: B. bacilliformis DNA from culture extracted by thermal lysis (100°C, 10 min.) using 16S 23S primers (positive control). 2: Whole blood extraction from an acute phase patient, using 16S 23S primers. 3: Whole blood extraction from an acute phase patient, using primers for Citrate Synthetase gene. 4: B. bacilliformis DNA from a culture extraction using primers for Citrate Synthetase gene. Base pairs 1500 bp 600 bp Molecular technics M 1 2 3 4

37. Chronic Phase: Peruvian wart (Verruga Peruana) Mularlesions

38. Chronic Phase: Peruvian wart (Verruga Peruana) Miliary lesions

39. Chronic Phase: Peruvian wart (Verruga Peruana) Miliary lesions with overwhelming infection

40. Chronic phase: some numbers The diagnostic tests in this phase are blood culture (13% of patients with verruga have bacteriemia), culture of the verrugous warts and Immunoblot with a sensitivity of 70% and specificity of 100% The diagnostic tests in this phase are blood culture (13% of patients with verruga have bacteriemia), culture of the verrugous warts and Immunoblot with a sensitivity of 70% and specificity of 100% The IFA has a sensitivity of 82% and specificity of 92% The IFA has a sensitivity of 82% and specificity of 92%

41. Immunity and infection One factor that complicates the clearance of the bacterium is that intra- erythrocytic Bartonella are protected from both humoral and cellular immune responses due to a lack of major histocompatibility complex (MHC) molecules on the surface of the mature erythrocytes One factor that complicates the clearance of the bacterium is that intra- erythrocytic Bartonella are protected from both humoral and cellular immune responses due to a lack of major histocompatibility complex (MHC) molecules on the surface of the mature erythrocytes They are unable to present antigens of their invaders to the immune system They are unable to present antigens of their invaders to the immune system