Hypothesis

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Compatibility Conundrums

• Why does a pathogen cause disease in some cases
  but not others?
• What determines whether microbes are commensals
  and/or opportunists?
• Do host-pathogen relationships evolve towards
  stability or extremes?

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Commensalism vs Persistence

• Commensals microbes that are part of the normal
  flora, not pathogenic (in theory?)
• Opportunists - microbes that cause disease under
  certain circumstances.

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Persistent Pathogens

• Cause disease initially host adaptive immune
  response keeps pathogen under control but cant
  clear all pathogens.
• Mycobacterium, Salmonella, Helicobacter
• and/or
• Cause disease later on host asymptomatic
  carriage at first, but disease potential later.
• Streptococcus, Neisseria, Haemophilus

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Strategies for Persistence

• Incomplete clearance by host
• Hide out (e.g. intracellular location)
• Virulence factors
• Antiphagocytic capsules
• Immunoglobulin proteases
• Antigenic variation of outer membrane proteins
• Colonize deeper tissues for reseeding

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Mycobacterium tuberculosis

• Tuberculosis often characterized by granulomatous
  lesions with necrotic centers.
• 1/3 of global population infected.
• Pulmonary exposure, bacillaemia, systemic spread
  to lymph nodes, adaptive immune response attempts
  to control infection.

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Persistent Mycobacteria

• Granulomas contain macrophages, T cells, B cells,
  dendritic cells, neutrophils, fibroblasts, and
  matrix components walled off from the body with
  epitheloid cells.
• Are bacteria in dormant state in granulomas or is
  continuous bacterial replication balanced by host
  immune response killing?
• Survival within macrophages may involve
  restricting phagolysosome fusion early on, and
  adapting to the phagolysosome environment later
  in infection.

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Salmonella enterica serovar Typhi

• Can cause Typhoid Fever or be asymptomatic. 1-6
  of infected people become chronic carriers.
• Salmonella infect intestinal Peyers patches,
  then penetrate epithelial barrier to infect
  phagocytes in lamina propria. Can be
  self-limiting or lead to systemic spread.

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Salmonella persistence strategies

• Intracellular infections
• Type III Secretion Systems
• Manipulation of apoptosis
• Systemic bacterial spread
• Periodic reseeding of mucosal surfaces

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Salmonella enterica serovar Typhi
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Helicobacter pylori

• An amazingly persistent pathogen considering the
  host defenses in the stomach that include acidic
  environs, mucus, gastric motility, and epithelial
  shedding!
• May cause asymptomatic infections, superficial
  gastritis, or cancer. Extremes are not conducive
  to Helicobacter survival.
• Predominantly extracellular lifestyle, though
  intracellular mode also observed.

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Helicobacter persistence strategies

• Evasion of innate responses
• arginase interferes with Nitric Oxide production.
• Cag PAI (TFSS) can help Helicobacter with
  megasome formation and blocking its own uptake
  by macrophages.
• Evasion of adaptive responses
• VacA interferes with antigen dependent T cell
  proliferation.

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Helicobacter persistence strategies

• Suppression of host inflammatory response
• LPS has low biological activity
• Flagellins have reduced ability to activate TLR5
• Genetic diversity
• Recombination common
• Phase variation in LPS
• Natural competence for DNA uptake
• Repopulation of the stomach

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Human- Wildlife - Domestic Animal Interface
Protozoal Problems Emerging at the

• P.A. Conrad, M. Miller, A. Kjemtrup, W. Miller
  and I.A. Gardner

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Theileria parva infects and transforms lymphocytes
Transformation without antigenic stimulation
or exogenous growth factors with continuous
proliferation clonal expansion metastatic
phenotype
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Tick Transmitted Protozoal Parasite
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Food Animal Production Cattle
Tick-transmitted Disease East Coast
Fever Theileria parva
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Vaccination against ECF

• Infection Treatment Method
• Inoculate tick-derived sporozoites
• Treat with tetracyclines
• Experimental studies with homologous challenge
  encouraging
• Field trials gt Not so!

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Early ECF Vaccination Trial in Ngong Hills, Kenya
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Discover

• Buffalo are assymptomatic carriers of multiple
  Theileria strains transmissible to cattle.

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Wildlife are good reservoirs of piroplasm
parasites
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Discover

• Looks can be deceiving gt
• morphologically identical parasites may be
  molecularly distinct
• different species or strains

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Always QuestionMicrobe identityHost
specificity
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SOUTHERNSEA OTTER(Enhydra lutris nereis)  
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SIGNS OF ENCEPHALITIS

• Muscle Tremors/Ataxia
• Paresis
• Seizures
• Inability to Prehend Food
• Visual Deficits

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8.5 of total sea otter mortality was attributed
to protozoal encephalitis. (Thomas et al,
1996)
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Protozoal Encephalitis in Southern Sea Otter
Doctoral Research of Melissa Miller and Chris
Krueder
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METHODS OF PARASITE CHARACTERIZATION

• Immunohistochemistry
• Parasite isolation in cell culture
• Growth/ motility in vitro
• Antigenic characterization
• Ultrastructure
• DNA amplification and sequence analysis

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Toxoplasma gondii
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Sarcocystis neurona
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DUAL BRAIN INFECTIONS! Toxoplasma gondii
Sarcocystis neurona
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Protozoal Brain Infectionsin California Sea
Otters(1998-2001)

• gt36 of fresh dead sea otters are T. gondii
  -positive at the time of necropsy
• Fresh otter mortality
• 16 Toxoplasma gondii
• 7 Sarcocystis neurona

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Relationship between protozoa of otters,
terrestrial animals,and humans?
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Toxoplasmosis in Humans

• Most asymptomatic or flu-like illness
• 10-30 of people in USA infected
• Greatest risk during pregnancy
• miscarriage or abortion
• congenital disease - blindness, retardation
• Severe/fatal encephalitis with AIDS

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Equine Protozoal Myeloencephalitis (EPM)
Sarcocystis neurona

• Progressive Asymmetric Nerve Damage
• Limb ataxia
• Muscle atrophy
• Cranial nerve dysfunction

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PROTOZOAL ENCEPHALITISA CASE OF PATHOGEN
POLLUTION?
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Role of freshwater runoff?
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Risk factors for protozoal infection?
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Epidemiologic Study

• Live and dead southern sea otters
• Identify risk factors for T. gondii exposure
• Examined three major categories of risk factors
• -Demographic
• -Geographic
• -Environmental
• Miller et al. 2002, International Journal for
  Parasitology 32997-1006

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ENVIRONMENTAL RISK FACTORS

• Coastal human population density
• (US 2000 census)
• Exposure to major municipal sewage outfalls
  (CCRWCQB)
• Exposure to surfacewater runoff (CCRWCQB)

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Logistic Regression Spatial/ Environmental
Factors
Risk Adjusted Factor odds ratio 95 CI
P____ Freshwater light 1.00 outflow
medium 1.07 0.48-2.4 0.876 exposure heavy
2.90 1.21-6.9 0.017 Sampling other
sites 1.00 location Morro Bay 9.31 2.26-38.31 0.0
02 ______________________________________________
__ Hosmer-Lemeshow goodness of fit statistic
0.96
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HYPOTHESIS Marine bivalves filter and
concentrate Toxoplasma oocysts 
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Success Depends on Teamwork!!
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