Prevalence of Giardia Cysts and Cryptosporidium Oocysts and Characterization of Giardia spp' Isolate

1
Prevalence of Giardia Cysts and Cryptosporidium
Oocysts and Characterization of Giardia spp.
Isolated from Drinking water in Canada.

• P.M. Wallis, S.L. Erlandsen, J.L Isaac-Renton,
  M.E. Olson,W.J. Robertson, and H. Van Keulen.

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BACKROUND

• Waterborne pathogens can pose threats to drinking
  water supplies, recreational waters, as well as
  source waters for agriculture and aquaculture.
• The probability of contracting giardiasis and
  cryptosporidosis from drinking water in Canada is
  not well understood. In order to obtain more
  accurate data, this study was carried out to
  determine the prevalence and potential for human
  infectivity.

3
BACKROUND

• These parasites can give otherwise healthy people
  diarrhea, headaches, nausea, cramps and a mild
  fever. But for people with immune problems, like
  cancer, HIV and AIDS, the condition is far more
  serious, possibly even fatal.

4

• Ultimately, the prevalence of waterborne
  protazoan infections depend on its concentration
  (dose), viability, and virulence against humans
  (pathogenicity).
• The purpose of this study was to measure these
  properties in Giardia isolates collected from
  Canadian drinking water samples.

5

• There have been several outbreaks of Giardiasis
  and Cryptosporidosis (much fewer) in British
  Columbia, Alberta, Ontario, Quebec, New
  Brunswick, and Newfoundland.
• Populations can be re-infected at sporadic levels
  depending on raw water quality, so its essential
  that the concentrations and pathogenicity of
  these parasites are obtained.

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METHODS

• SAMPLE COLLECTION
• SAMPLE ANALYSIS IDENTIFICATION OF GIARDIA AND
  CRYPTOSPORIDIUM.
• VIABILITY TESTING FOR GIARDIA CYSTS
• Pulsed Field Gel Electrophoresis (PFGE)
• AMPLIFICATION OF ISOLATE DNA USING
  PCR-RECOMBINANT PROBE TESTING

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Drawbacks

• Variations in turbidity, organic matter, and
  algal concentration all affected cyst recovery
  efficiency.
• Many of the cysts in the water samples are not
  recovered even by the best available detection
  methods. This leads to underestimation of the
  true presence of the parasite and also makes it
  difficult to detect viability of giardia.
• All isolates recovered by in vitro or in vivo
  techniques are clones and may not be
  representative of all the cysts present in the
  water. Recognizing this difficulty, they
  attempted to evaluate pathogenicity by recovering
  Giardia isolates from gerbils and comparing them
  to strains recovered from raw sewage (presumably
  human in origin) by using pulsed-field
  electrophoresis, DNA karyotyping, isoenzyme
  analysis, and hybridization of DNA with
  recombinant DNA probes.

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SAMPLE COLLECTION

• Samples were collected from 72 sites across
  Canada at monthly intervals for 4 years.
• 24 samples from each site was collected.
• All the sites obtained their raw water from
  surface waters.
• 58 of the 72 site samples treat their water by
  chlorination alone, without any filtering.
• The remaining 14 employed some type of
  filtration.
• In total, 1,760 samples were collected. Of this
  1,173 were from raw water, 423 treated drinking
  water, and 164 were raw sewage samples.
• The sample collection device was constructed in
  their laboratory. It was composed of a hose,
  filters, and a pressure reducer. All components
  in contact with water were brass, plastic, or
  stainless steel.

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Sewage Sample Collection

• Sewage samples were collected in 500ml containers
  in a composite sampler.
• 200ml of sewage was concentrated by
  centrifugation into a pellet and stained as
  described earlier without further processing.
• The resulting pellet was split between
  immunofluorescence analysis and gerbil infection.

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WATER SAMPLE ANALYSIS

• Samples were washed with phosphate buffered
  saline (PBS) between the application of the
  primary antibody and the fluorescein
  isothiocyanate (FITC) labeling antibody.
• Incubation
• Pellets washed with PBS then with deionized water
  before spotting on a window slide.
• Slides were scanned under oil and identification
  was confirmed at 1000X by
• Shape general spherical shape folding
• in the cyst wall.
• Size usually 4-6 micrometers
• Strength of Immunofluorescence usually 50 of
  that seen in controls a distinct fluorescence
  around the wall.

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Viability Testing of Giardia using PI Method

• Viability was tested by treating water and sewage
  samples with flourescein diacetate with propium
  iodide (PI). A portion of this was used to infect
  gerbils.
• PI is the most ideal method to detect viability
  due to the low cyst counts.
• PI will not penetrate an intact cyst wall, so
  its absence indicates a living cyst.
• Gerbils were pretreated with 3-5 doses of
  metronidazole (Flagyl). Despite treatment with
  antibiotic, they were still infected. They were
  sacrificed 7 days after inoculation.
• Infection was confirmed or rejected by examining
  biopsy snips of the small intestine for
  trophozoites. Trophozoites were then cultured and
  used in Isoenzyme analysis, PFGE, and DNA
  amplification using PCR.

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Isoenzyme analysis

• If trophozoites were present, they were stripped
  from the gut and cultured.
• Isolates from culture were washed 3 times with
  PBS.
• Equal volumes of EDTA, dithioreitol, and alpha-
  aminohexanoic acid were added to the
  trophozoites. They were left overnight at -135
  degrees C, thawed to lyse the organism,
  centrifuged at 4 degrees C.
• Lysates of the different organisms were applied
  to different wells in the gel surface and
  electrophoresed.
• Different gels were used for each enzyme system
  tested.

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Pulsed Field Gel Electrophoresis (PFGE)

• Sample preparation
• Trophozites from selected isolates were cultured
  then incubated at 37 degrees C.
• Tropgozoites were harvested by chilling and
  centrifuging.
• Pellets were washed twice in PBS and TSE , then
  counted in a neubauer chamber.
• Equal volumes of trophizoites and low melting
  point 1 agarose were combined. They were then
  added to the wells and allowed to solidify.
• Enzymes were added to the well to digest away the
  cell wall and proteins and incubated.
• After a week of electrophoresis, the gels were
  removed, washed in buffer, and stained with
  ethidium bromide.

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Microscopy Results Water and Sewage Samples.

• Highest proportion of the parasites was found in
  raw sewage(72.6), followed by raw (20.9) and
  drinking (18.2) water.

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Prevalence in Raw and Drinking Water Samples.

• Fig 1 Giardia cysts were most frequently found
  in New Brunswick, Nova Scotia, and Newfoundland.
  These sites treat their water by chlorination
  alone, with no filtration and minimal contact
  time before water reaches the customer (5-30
  min).
• Cryptosporidium oocysts were found much less
  frequently. This may be because none of the
  sample sites were situated downstream of
  agricultural areas or sewage effluents.

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Prevalence in Raw Sewage Samples

• The prevalence of these parasites were much
  higher in raw sewage samples. They usually
  contained more than 1,000 cysts per liter
• Cryptosporidium (6.2) was much less prevalent
  than giardia. (72.6). This confirms that
  giardiasis is a common enteric parasite in
  Canada.
• Sewage monitoring is a useful indicator of the
  prevalence rate in a community.

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Results Viability Testing of Giardia

• Of the 167 positive drinking water and sewage
  samples treated with PI, the average viabilities
  observed were
• 24.6 drinking water
• 38.9 sewage samples

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Infection results of Gerbil Inoculation

• The rate of giardia infection in gerbils was
• 2.2 in raw water
• 7.6 in treated water
• 22.2 in sewage
• The difference in infection rates of sewage and
  water probably reflects dosage, which is 3.5
  times higher in sewage.

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Isolated Giardia Strains
A total of 13 isolates were established in
culture. The viability was measured from 33
samples, and the rate of infection was 8 in
water samples and 32 for sewage.
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Isoenzyme Analysis Results

• 3 distinct zymodeme groups were observed. Group
  one isolates were both waterborne outbreak
  strains from Newfoundland, but groups 2 and 3
  included isolates from diverse geographic
  sources.
• Water source isolates were found in each group,
  but zymodeme grouping was not predictive of
  either geographic source or type of specimen.

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PFGE RESULTS

• Banding patterns of chromosomes were
  reproducible, and four or five bands were
  observed in the set of isolates tested.
• Karyotypes were identified on the basis of
  estimated size, number, and spatial position. The
  isolates could be divided into 3 groups on the
  basis of karyotype.

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• High concentrations of cysts in raw sewage in
  combination with positive results from drinking
  water are suggestive and should be cause for
  concern.
• The apparent existence of giardia cysts in sewage
  is clear warning against the discharge of
  untreated sewage to any water that may be a
  potable water source or inhabited by aquatic
  mammals (beavers muskrats). Establishment of
  human infective giardia in wild animal reservoirs
  helps to spread the parasite through animal
  migration. This may magnify a low level of
  contamination to potentially dangerous
  concentrations.

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• The comparison of cyst concentrations between
  outbreak and non-outbreak communities is the
  basis for a proposed action level for giardia
  cysts.
• An action level of 3-5 cysts / 100 liters was
  proposed on the basis of monitoring data from
  outbreak situations.
• In this study, average giardia cyst
  concentrations above 3-5 cysts per 100 liters in
  drinking water were always associated with high
  levels of giardiasis in the communities to which
  it was supplied. Giardia cysts were most
  frequently found in New Brunswick, Nova Scotia,
  and Newfoundland. These sites treat their water
  by chlorination alone, with no filtration and
  minimal contact time before water reaches the
  customer (5-30 min). There are presently about
  1,000 communities in Canada that employ this
  method alone and they are the greatest at risk
  for outbreaks.

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• The isolates available for isoenzyme, PFGE
  karyotyping, and PCR- recombinant probe analysis
  proved to be heterogeneous.
• Isolates of giardia are heterogenous, with both
  heritable differences between genetic groups and
  surface antigenic variation. Antigenic variation
  is likely to be involved in determining the
  clinical spectrum of giardiasis and its ability
  to reinfect. This is why chronic infections are
  common with this disease.
• These methods cannot distinguish between
  isolates that are infective to humans and those
  that are not. The source of the waterborne
  strains is unknown, but the sewage strains are
  presumably human in origin and are most probably
  human infective.
• Although these results are inconclusive
  concerning human pathogenicity, the strains do
  fall into distinct groups as predicted. The
  origin of all the isolates in this study suggests
  that all these isolates are potentially infective
  to humans.