BartonellaBartonella

Prepared by:Prepared by:

Ohood R. SarsourOhood R. Sarsour

IntroductionIntroduction Bartonella species (formerly known as Bartonella species (formerly known as

Rochalimaea)Rochalimaea) Linked to a number of emerging zoonotic Linked to a number of emerging zoonotic

diseasesdiseases B.quintana, B.bacilliformis, B.henselaeB.quintana, B.bacilliformis, B.henselae

Unique and common feature is ability to cause Unique and common feature is ability to cause vasoproliferative lesions (process of pathological vasoproliferative lesions (process of pathological angiogenesis)angiogenesis)

Infection affects function of endothelial cells Infection affects function of endothelial cells resulting in cell invasion, suppression of resulting in cell invasion, suppression of apoptosis and induction of proliferation, apoptosis and induction of proliferation, resulting in vasoproliferative tumor growth resulting in vasoproliferative tumor growth

B. henselaeB. henselae Gram negative aerobic rodsGram negative aerobic rods Main reservoir is catsMain reservoir is cats

Transferred to humans through scratches and Transferred to humans through scratches and bites (fleas/ticks??)bites (fleas/ticks??)

Resulting illness is Cat Scratch Disease Resulting illness is Cat Scratch Disease (CSD)(CSD)

About 40% of cats carry About 40% of cats carry B.henselaeB.henselae Cats are asymptomatic, although Cats are asymptomatic, although

bacteria can be cultivated from their bacteria can be cultivated from their blood for up to 2 yearsblood for up to 2 years This persistent infection due to persistence in This persistent infection due to persistence in

red blood cellsred blood cells

EpidemiologyEpidemiology

Cases of CSD occur worldwideCases of CSD occur worldwide Higher incidence of Bartenellosis in Higher incidence of Bartenellosis in

warmer and more humid areas of the warmer and more humid areas of the United StatesUnited States More fleas and ticksMore fleas and ticks

Children 2-24 and veterinarians make Children 2-24 and veterinarians make up 80-90% of human casesup 80-90% of human cases

22 000 cases of CSD are diagnosed 22 000 cases of CSD are diagnosed per year in the United Statesper year in the United States Most cases occur in late fall and winterMost cases occur in late fall and winter

InfectionsInfections

CSD is a self-limiting disease characterized CSD is a self-limiting disease characterized by lymphadenopathy and infection at the by lymphadenopathy and infection at the site of injurysite of injury

Lasts 6-12 weeks in healthy individuals and Lasts 6-12 weeks in healthy individuals and can be accompanied by headaches, nausea, can be accompanied by headaches, nausea, vomiting and fatiguevomiting and fatigue

Severity of CSD depends on immune status Severity of CSD depends on immune status of the hostof the host

Immunocompromised individuals can Immunocompromised individuals can develop Bacillary angiomatosis (BA) and develop Bacillary angiomatosis (BA) and Parinaud’s oculoglandular syndrome (POS)Parinaud’s oculoglandular syndrome (POS)

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

Virulence FactorsVirulence Factors

VEGF (vascular endothelial growth factor)VEGF (vascular endothelial growth factor) Triggers angiomatosis and has a pathogenic role Triggers angiomatosis and has a pathogenic role

in vasoproliferative disorders (such as BA)in vasoproliferative disorders (such as BA) Highly mitogenic to endothelial cellsHighly mitogenic to endothelial cells B.henselae B.henselae induces host cell production of VEGFinduces host cell production of VEGF This production and subsequent proliferation is This production and subsequent proliferation is

necessary for angiogenesis and BAnecessary for angiogenesis and BA This proliferation also promotes growth of This proliferation also promotes growth of

B.henselae’sB.henselae’s own cells own cells

PiliPili Expression of pili allows adhesion and Expression of pili allows adhesion and

invasion of host cellsinvasion of host cells Pili are also thought to be responsible Pili are also thought to be responsible

for the induction of VEGF as pilifor the induction of VEGF as pili- - mutants mutants have a significant decrease in have a significant decrease in production of VEGFproduction of VEGF

Type 4 secretion system (Type 4 secretion system (virB-D4virB-D4)) Consists of a multiprotein channel that Consists of a multiprotein channel that

allows transportation of DNA or protein allows transportation of DNA or protein into the host cellinto the host cell

virBvirB operon encodes 10 genes, 8 of which operon encodes 10 genes, 8 of which known to encode a T4SSknown to encode a T4SS

virD4virD4 locus located just downstream locus located just downstream virB5virB5 encodes a 17kD protein found in encodes a 17kD protein found in

patients with CSDpatients with CSD Function not known but not found Function not known but not found

outside outside BartonellaBartonella species species

Downstream region of Downstream region of virBvirB encodes Bep encodes Bep proteins that are translocated into the proteins that are translocated into the endothelial cellsendothelial cells These proteins thought to mediate all These proteins thought to mediate all virB-virB-

virD4-virD4-dependent changes of endothelial cell dependent changes of endothelial cell function function

Recent studies have shown that a Recent studies have shown that a mutation in either mutation in either virBvirB or or virD4virD4 prevents prevents intraerythrocytic infection as well as intraerythrocytic infection as well as endothelial cell invasionendothelial cell invasion

Type 4 Secretion System (Type 4 Secretion System (TrwTrw)) Second T4SS identified that is active Second T4SS identified that is active

during endothelial cell infectionduring endothelial cell infection Primary function to establish contact Primary function to establish contact

with erythrocytes through surface with erythrocytes through surface expressed T4SS piliexpressed T4SS pili

DeforminDeformin Hydrophobic secreted protein Hydrophobic secreted protein Affinity for albuminAffinity for albumin Formation of pits and trenches on red Formation of pits and trenches on red

blood cell membranes that aids in blood cell membranes that aids in colonization and entry to the cellcolonization and entry to the cell

Outer Membrane Proteins (OMPs)Outer Membrane Proteins (OMPs) Induce NFInduce NFκκB-dependent upregulation of B-dependent upregulation of

E-selectin and ICAM-1E-selectin and ICAM-1 Leads to increased adhesion molecule Leads to increased adhesion molecule

expressionexpression NFNFκκB plays a role in initiation and B plays a role in initiation and

regulation of the body’s regulation of the body’s proinflammatory responseproinflammatory response

Persistent InfectionPersistent Infection

Endothelial cell interactionEndothelial cell interaction Primary nichePrimary niche Most information gathered using HUVEC Most information gathered using HUVEC

cellscells Bartonella Bartonella species can enter endothelial species can enter endothelial

cells by rearranging actin cytoskeletoncells by rearranging actin cytoskeleton B.henselaeB.henselae can also enter using can also enter using

“invasome” mechanism“invasome” mechanism Bacterial aggregate formation on cell surface Bacterial aggregate formation on cell surface

that is engulfed and internalized through an that is engulfed and internalized through an actin-dependent processactin-dependent process

Intraerythrocytic persistenceIntraerythrocytic persistence Common theme of Bartonella is prolonged Common theme of Bartonella is prolonged

intraerythrocytic bacteremia associated with intraerythrocytic bacteremia associated with transmission by blood-sucking arthropodstransmission by blood-sucking arthropods

Adherence mediated by pili and OMPs Adherence mediated by pili and OMPs exposed on bacterial surfaceexposed on bacterial surface

Exact mechanism is not known but assumed Exact mechanism is not known but assumed to be bacterial-induced endocytosisto be bacterial-induced endocytosis

Assisted by deformin secretionAssisted by deformin secretion

After invasion intracellular multiplication occursAfter invasion intracellular multiplication occurs Limited by quorum sensing to prevent Limited by quorum sensing to prevent

hemolysishemolysis Bacteria survive for the duration of the cell life Bacteria survive for the duration of the cell life

and remain in the blood stream for several weeks, and remain in the blood stream for several weeks, facilitating transmissionfacilitating transmission

Bacteria seeded into blood stream every 5 daysBacteria seeded into blood stream every 5 days Unique persistence is an adaptation to mode of Unique persistence is an adaptation to mode of

transmissiontransmission Intraerythrocytic bacteremia subsides due to Intraerythrocytic bacteremia subsides due to

strong IgG antibody response (in animal models)strong IgG antibody response (in animal models)

Prevention and Prevention and TreatmentTreatment

Proper cat careProper cat care Keeping cats indoorsKeeping cats indoors Using flea productsUsing flea products Prevent roughhousing with kittensPrevent roughhousing with kittens Do not let cats like open woundsDo not let cats like open wounds Wash cat wounds out with warm waterWash cat wounds out with warm water

Most healthy people recover without Most healthy people recover without treatment in anywhere from 3 weeks treatment in anywhere from 3 weeks to several monthsto several months

Immunocompromised individuals Immunocompromised individuals may develop more serious may develop more serious complications and possibly deathcomplications and possibly death

If antibiotics are prescribedIf antibiotics are prescribed Erythromycin, rifampin, doxycyclineErythromycin, rifampin, doxycycline

Antibiotics can not reach Antibiotics can not reach intraerythrocytic bacteria but can intraerythrocytic bacteria but can prevent new waves of parasitismprevent new waves of parasitism

A recent study has shown that cats A recent study has shown that cats treated for bacteremia caused by treated for bacteremia caused by B.henselae were resistant to B.henselae were resistant to reinfection when challenged reinfection when challenged following recoveryfollowing recovery

B.quintanaB.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 mediaThe bacterium can be grown on axenic media When grown on blood agar,rough colonies When grown on blood agar,rough colonies

embedded in the agar obtained after 12to14 embedded in the agar obtained after 12to14 daysdays

Humans are the reservoir of the Humans are the reservoir of the bacterium,and the human body louse is its bacterium,and the human body louse is its usual vectorusual vector

The bacterium has atropism for endothelial The bacterium has atropism for endothelial cells,leading to angioproliferative cells,leading to angioproliferative lesions,observed in bacillary angiomatosis.lesions,observed in bacillary angiomatosis.

TransmissionTransmission

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

Clinical ManifestationsClinical Manifestations

Trench Fever.Trench Fever. Chronic Bacteremia.Chronic Bacteremia. Endocarditis.Endocarditis. Bacillary Angiomatosis.Bacillary Angiomatosis. Lymphadenopathy.Lymphadenopathy.

DiagnosisDiagnosis

Serologic testsSerologic 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), allantibodies disappear. After adsorption with B. henselae antigen(C), antibodies against B. quintana (1) persist. This reaction showsB. quintana infection.

Culture.Culture. Molecular Biology.Molecular Biology. ImmunohistochmisImmunohistochmis

try and try and ImmunoflurescencImmunoflurescence.e.

. Immunohistochemical

demonstration of Bartonella sp Laser confocal microscopy showing the intraerythrocyticlocation of Bartonella quintana.

Antimicrobial drug Antimicrobial drug susceptibility of B. susceptibility of B.

quintanaquintana Evaluation of susceptibilities to Evaluation of susceptibilities to

antimicrobial drugs has been performed antimicrobial drugs has been performed in both axenic media and cell culture.in both axenic media and cell culture.

Penicillines,cephalosporins,aminoglycosiPenicillines,cephalosporins,aminoglycosides,chloramphenicol,tetracyclines,rifamdes,chloramphenicol,tetracyclines,rifampin,fluoroquinolones,and cortimonazole.pin,fluoroquinolones,and cortimonazole.

However only aminoglycosides have However only aminoglycosides have bactericidal effect.bactericidal effect.

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.

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

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

PathogenesisPathogenesis Bartonella bacilliformisBartonella bacilliformis is transmitted is transmitted

by the bite of the suspected vector by the bite of the suspected vector Lutzomyia sppLutzomyia spp

Following transmission, the Following transmission, the bacterium infects red blood cells and bacterium infects red blood cells and endothelial cellsendothelial cells

The physical damage and The physical damage and introduction of antigens in the introduction of antigens in the membranes of the red cells stimulate membranes of the red cells stimulate the Reticuloendothelial System to the Reticuloendothelial System to produce an intense produce an intense erythrophagocytosis by macrophages erythrophagocytosis by macrophages and histiocytic cells resulting in and histiocytic cells resulting in severe extra vascular hemolytic severe extra vascular hemolytic anemiaanemia

The diseaseThe disease

The clinical symptoms of The clinical symptoms of bartonellosis are pleomorphic and bartonellosis are pleomorphic and some patients may be asymptomatic some patients may be asymptomatic

The two classical clinical The two classical clinical presentations are the acute phase presentations are the acute phase and the chronic phase, and the chronic phase, corresponding to the two different corresponding to the two different host cell types invaded by the host cell types invaded by the bacteriumbacterium

Acute phase: Oroya fever Acute phase: Oroya fever or Carrion’s diseaseor Carrion’s disease

The mean incubation time is 21 days The mean incubation time is 21 days (range 10 to 270 days)(range 10 to 270 days)

The diagnostic tests in this phase are:The diagnostic tests in this phase are:

Diagnostic test Sensitivity SpecificityBlood smear 36-73 91-96

Immunoblot 70 94

PCR(16S-23S) 47 98

Values in porcentaje

The diagnosisThe 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.

Lane A: Positive control poolLane Band C: Bartonella bacilliformis-positive serum taken from a patient in acute phaseLane D: Negative control pool

Immunologic technics: Immunologic technics: Sonicated immunoblotSonicated immunoblot

A B C D

20 kDa18 kDa17 kDa14 kDa

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 technicsMolecular technics

M 1 2 3 4

Chronic Phase: Peruvian Chronic Phase: Peruvian wart (Verruga Peruana)wart (Verruga Peruana)

Mularlesions

Chronic Phase: Peruvian Chronic Phase: Peruvian wart (Verruga Peruana)wart (Verruga Peruana)

Miliary lesions

Chronic Phase: Peruvian Chronic Phase: Peruvian wart (Verruga Peruana)wart (Verruga Peruana)

Miliary lesions with overwhelming infection

Chronic phase: some numbersChronic phase: some numbers

The diagnostic tests in this phase The diagnostic tests in this phase are blood culture (13% of are blood culture (13% of patients with verruga have patients with verruga have bacteriemia), culture of the bacteriemia), culture of the verrugous warts and Immunoblot verrugous warts and Immunoblot with a sensitivity of 70% and with a sensitivity of 70% and specificity of 100% specificity of 100%

The IFA has a sensitivity of 82% The IFA has a sensitivity of 82% and specificity of 92%and specificity of 92%

Immunity and infectionImmunity and infection

One factor that complicates the One factor that complicates the clearance of the bacterium is that clearance of the bacterium is that intra-erythrocytic intra-erythrocytic BartonellaBartonella are are protected from both humoral and protected from both humoral and cellular immune responses due to a cellular immune responses due to a lack of major histocompatibility lack of major histocompatibility complex (MHC) molecules on the complex (MHC) molecules on the surface of the mature erythrocytes surface of the mature erythrocytes

They are unable to present antigens of They are unable to present antigens of their invaders to the immune systemtheir invaders to the immune system