The ability of tetracyclines to treat Lymphatic Filariasis by killing the endosymbiotic bacteria Wolbachia

1
The ability of tetracyclines to treat Lymphatic
Filariasis by killing the endosymbiotic bacteria
Wolbachia

• Rajan, T.V 2004. Relationship of Anti-microbial
  activity of Tetracyclines to their ability to
  block the L3 to L4 molt of the human filarial
  parasite Brugia malayi. American Journal of
  Tropical Medicine 7 24-28.
• Hannah Dowling
• BIO 475

2
Contents

• Lymphatic Filariasis
• Physiological Effects on humans
• Mosquito life cycle
• Treatments
• Tetracyclines - Doxycycline
• Conclusion

3
Lymphatic Filariasis

• Affects over 120 million people worldwide
• Found mainly in the tropics of Africa, Asia, and
  Central America
• Causes severe disabilities and lymphatic system
  destruction

4
Physiological manifestations

• Lymphedema, elephantiasis, hydrocele, and scrotal
  elephantiasis
• The extremities can swell to several times their
  normal size, which, along with decreased
  lymphatic function, can lead to bacterial
  infections that the body cannot fight effectively

5
Diagnosis

• Microfilaremia levels can be detected in the
  blood only after prolonged infection by blood
  smear
• The blood sample must be taken in accordance with
  the known periodicity of the microfilariae to
  achieve the most accurate results
• Microfilariae are present in high numbers at
  night, when chances of transmission are high
  because of night-biting mosquitoes

6
Transmission and Prevention

• Transmitted by Culex, Anopheles, and Aedes
  Mosquitoes through a complex life cycle
• Larvae in human ingested by mosquito, larvae
  mature and migrate to proboscis of mosquito, and
  move to new human host at mosquitos next blood
  meal
• Transmission prevented by use of mosquito spray
  (DEET) and bed nets

7
Filariae

• Filariae Brugia malayi (limited to Asia),
  Wuchereria bancrofti (tropical areas worldwide),
  and Brugia timori (Indonesia)
• 10cm long and measure 2cm in width
• Can survive for up to 10 years and begin to
  reproduce approximately 6 to 12 months after
  infection

Wuchereria bancrofti in a blood smear
8
Wolbachia

• Endosymbiotic bacteria found in up to 70 of
  arthropods
• In filariae, they are constrained to the
  organisms hypodermis and the female reproductive
  tissues
• Transmitted vertically
• Gram-negative
• Genome contains 1.1 million neucleotides

9
Wolbachia Bacteria
(Left) Cross-Section of a Female Worm from an
Extirpated Nodule from a Patient with
Onchocerciasis Wolbachia, endosymbiotic bacteria
of filarial nematodes important for embryo
development, are targets for antifilarial
treatment. The bacteria here were stained with
antibodies against Yersinia Hsp60. The bacteria
are located in the hypodermis and oocytes of the
worm (arrows). h, hypodermis i, intestine m,
muscle u, uterine tube. (Right) Cross Section of
Wuchereria bancrofti, with arrows showing
Wolbachia.
10
Wolbachia

• Genome contains genes necessary to make one
  nucleic acid and all necessary genes to infect
  new hosts and evade the hosts immune system
• Lacks genes to produce lipopolysacharride but
  produces inflammatory reaction via TLR4
• Has genes that are necessary for the production
  of riboflavin, flavin adenine dinucleotide, and
  heme
• Type IV secretion system

11
Treatment

• Wolbachia are present in the tissue responsible
  for synthesizing cuticle (the hypodermal
  syncitium) - may have a crucial role in the
  molting process of the filarial host
• Diethylcarbamazine (DEC), Ivermectin, and
  Albendazole have microfilaricidal effects but no
  adulticidal effects

12
Proportion of worms/larvae killed by the most
common treatments
(Data courtesy of Stolk et al., 2004)
13
Tetracycline (Doxycycline)

• Tetracycline inhibits the L3 to L4 molt of the
  larvae to completely stop reproduction
• After continued treatment, it has limited
  macrofiliaricidal effects
• More research is needed on the mechanism of
  effect and of the accompanying side effects

14
Chemically Modified Tetracyclines

• CMTs are tetracyclines that have had the
  diethylamino group attached to the fourth carbon
  of the A ring of the tetracycline molecule
  removed
• This action removes the antimicrobial activity
  but has higher macrofiliaricidal effects than
  tetracyclines
• CMTs are more potent but may be available for
  mass treatment and are still being researched

15
Tetracycline vs. CMTs
At high concentrations (10-20µg/mL),
tetracycline completely blocked molting, but
viability of the larvae was not affected. This
means that the larvae are not molting but still
can be seen in constant motion which means they
are not being killed by tetracycline. In
experiments using Col-3 and Col-8 at the same
high concentrations (10-20µg/mL), the larvae were
killed within 24 hours. At much lower
concentrations, Col-3 was shown to inhibit
molting without interfering with the viability of
the larvae (concentrations of 0.3µg/mL). (Charts
courtesy of T.V. Rajan, 2004, The Lancet)
16
Amplification of Wolbachia DNA by PCR

• Lanes 1-4 contain Wolbachia DNA ribosomal
  amplified (rDNA), and lanes 5-8 contain Brugia
  malayi collagen 1
• Lanes 1,5 Medium
• Lanes 2,6 0.5µg/mL Col-3
• Lanes 3,7 1µg/mL Col-8
• Lanes 4,8 50µg/mL tetracycline

17
Results

• Results showed that the amounts of Wolbachia DNA
  stayed relatively the same between the control
  group and those treated with CMTs
• Although CMTs do not have anti-microbial
  properties, they are more potent than
  tetracyclines and may be more effective in mass
  administration

18
Conclusion

• More research is needed to find an appropriate
  method of treatment for this disease
• Tetracyclines such as Doxycyline look most
  promising because they are proven to be safe and
  effective, but may not be given to entire
  populations because of the risk of resistance

19
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20
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