SYTTY
MICROBIAL RISKS ASSOCIATED WITH DRINKING WATER: GIARDIA AND CRYPTOSPORIDIUM
Project leader: Marja-Liisa Hänninen, University of Helsinki,
Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine,
P.O. Box 57, FIN-00014 University of Helsinki, Helsinki, Finland, tel.
+358-9-19149704,
e-mail: Marja-Liisa.Hanninen@helsinki.fi
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Researchers:
Ruska-Rimhanen-Finne, Department of Food and Environmental Hygiene,
email: Ruska.Rimhanen-Finne@helsinki.fi
Ari Hörman, Defence forces, Finland
Consortium: Microbiological risks of drinking water contaminated
with protozoans, viruses or cyanobacterial toxins
Financing SYTTY organisation: Tekes
Funding from SYTTY / Total funding of project (€): 157701
/ 199848
Person-months of work funded by SYTTY / Total person-months of work:
38 / 55
KEY WORDS: Giardia, cryptosporidium, methods, raw water,
sewage, faecal samples
EXTENDEN ABSTRACT
1 Introduction
Giardia and Cryptosporidium are protozoan parasites causing diarrhoeal episodes in humans in developing as well as developed countries. Cryptosporidia have been identified in 1 - 4% of diarrhoeal patients in many western world countries and in 0.2 to 0.5% in asymtomatic carriers. 12 - 24% of faecal samples of HIV- infected patients with diarrhoea were positive for cryptosporidia. The incidence of Giardia infections in developed countries has shown to vary from 1 to 4 to % and the infection seems to rather common also in asymptomatic people. In Finland the number of annually reported cases to the National Registry for Infectious Diseases (NRID) is low for both organisms, approximately 300 – 400 giardia cases and only a few cryptosporidium cases A human patient with active infection may excrete 105 -107/g of cysts or oocysts.
Cryptosporium is a coccidian parasite and giardias are members of flagellates. The life cycles of giardia and cryptosporidia are similar. In the initial stage of infection, cysts or oocysts are ingested. In the intestines, excystation will occur and trophozoites (Giardia) or sporozoites (Cryptosporidium) are released. The host excretes cysts or oocysts in the environment. The size giardia cysts is 8 –12 µm x 7 -10 µm and that of oocysts of Cryptosporidium is approximately 4-6 µm. The cell wall of cysts and oocysts is covered by a chitin-like material which makes them very resistant against environmental conditions, including drinking water desinfection. In water environment cysts and oocysts will survive and be viable for months.
The taxonomy of Giardia or Cryptosporidium spp. is not clear. G. intestinalis is the species infective for humans. Many animal species have their host-specific giardia species which does not infect humans. Two genetic assemblages, A and B of G. intestinalis seem to have zoonotic transmission capacity. Similarly, C. parvum, species infecting humans does have two genotypes (genotype 1 and 2). Genotype 1 is strictly human associated and genotype 2 has several hosts including ruminants and humans. Genotype 2 has zoonotic transmission potential. Genotype 3 is colonising cats (C. felis) and canine genotype is 4. Genotypes 3 and 4 have also been isolated a few times from humans. C. andersonii colonises gastrointestinal tract of adult cattle. There are also several other Cryptosporidium species associated e.g., with birds (chicken, turkey), mice etc.
Giardia and Cryptosporidium have caused several waterborne outbreaks especially in the USA and United Kingdom. In these countries plenty of research, risk assessment and risk management efforts has been directed to the topic Giardia and cryptosporidium and drinking water. Drinking water treatment procedure should be able to remove 3 to 4 logs of both organisms from surface water if the risk for acquisition infection from drinking water is at acceptable level. In a study from USA 1000 - 10 000 cycst/oocysts per liter were counted in the primary effluent, and 10 cysts per liter and 17 oocysts per liter in the secondary effluent of sewage treatment plant. Effluent water from sewage treatment plant is an important source for raw water contamination.
The aim of our study was 1) to develop methods for studies of source and drinking water samples for giardia and cryptosporidium, 2) to apply these methods for studies of Finnish raw water sources to find the level of contamination, especially in areas where surface water is used as a source for drinking water production, 3) to study contamination sources of surface water, and 4) to develop methods for studies of animal faecal samples. This study belonged to a consortium including projects dealing with risks of cyanobacterial toxins and viruses on the safety of drinking water.
2 Materials and methods
Samples
A. Raw water samples: 10 l grab water samples were collected
from 33 river systems during spring 1999 – spring 2000 (a total of
88 samples): In addition 17 Environchek samples were studied from
three surface treatment plants. Autumn 2000 – autumn 2001 10 l grab samples
were collected four times from 22 sampling sites (a total of 142 samples).
In addition, 42 Envirochek samples (6 to 120 l) were collected from
9 surface water plants. B. In April-May 2000 raw sewage sludge samples
were collected from 12 municipal wastewater treatment plants and in April-May
2002 sludge samples were collected from 24 treatment plants. C. Intestinal
contents from 20 beavers, 20 calves with diarrhoea, 100 healthy cats and
500 healthy dogs.
Development of methods
Concentration methods
Three different methods were tested for concentration of cysts and
oocysts from raw water samples: a. a flocculation technique using CaCl2
as a flocculant for 10 l grab samples (collected Spring 1999 -Spring 2000)
(Vesey et al. 1993), b. membrane filtration technique (USEPA 1623, 1999)
through polycarbonate membrane (pore size 1.2 µm). After filtration
the samples were further concentrated and purified by immunomagnetic separation
technique (IMS, Dynal, Rimhanen-Finne et al. 2001, 2002), c. concentration
by filtration (Envirochek, Gelman) directly from the raw water inlet tube
at drinking water plant. The filters were sent to the Department of.Food
and Environmental Hygiene for further processing which included concentration
by polycarbonate filter and IMS. The final volume of the concentrate was
50 to 100 µl and the whole quantity of concentrate was studied (Rimhanen-Finne
et al. 2002).
A novel method, using biological concentration performed by mussels (Unio tumidus) was tested at Vantaa river autumn 2001. Fresh clean river mussels came from Keski-Suomen Ympäristökeskus (S. Herve) and they were returned into four places at Vantaa river in baskets which included always 10 mussels each. The baskets were collected four times between October - November 2001 and studied at laboratory for giardia and cryptosporidium by microscopy and PCR. At same time a conventional 10 l grab sample was also taken. Influent (10 ml) and effluent waste water (2 litre) samples were concentrated similar way as raw water samples. Raw sewage sludge samples: 1 g was directly concentrated and purified by IMS Faecal samples were concentrated for microscopic studies by ethylene acetate-formalin technique.
Immunofluorescence microscopy
For microscopic examination of the samples, aliquots of weighted samples
were spread onto wells on microscopic slides (Waterborne Inc, USA). After
drying and fixation of the samples, and the cysts and oocysts were
stained by combined monoclonal antibodies against C. parvum oocysts and
G. intestinalis cysts conjugated with FITC (Waterborne Inc). The slides
were incubated in moist chamber for 30 min at 37 C, washed with PBS (pH,
7.3) and looked under epifluorescence microscope and typically stained
cysts and oocysts were counted from the wells. Positive controls were always
included.
PCR methods
DNA from purified cysts and oocysts was released by 5 – 15 freeze-thaw
cycles. The primers tested for PCR amplification of Cryptosporidium were
SSU rRNA , cry9/cry15 (oocyst wall-protein), TRAP and LaxA/LaxB (Rimhanen-Finne
et al. 2001, 2002. The primers used for Giardia were GDH (glutamate
dehydogenase gene) and MAH (Rimhanen-Finne et al. 2001, unpublished
results) and the primers specific either for assemblage A or B. Sensitivity
and specificity of the primers were tested with spiked raw water,sewage
sludge and faecal samples.
Genotyping of Cryptosporidium samples
For genotyping PCR- RFLP (cry9/cry15) of samples positive in PCR for
C. parvum the PCR was performed. Genotyping of Giardia was
performed for samples positive with GDH primers by using PCR specific either
for Giardia assemblage A or B. The digested PCR products were run
on 3% agarose gels and the lengths of produced fragments were measured
and compared with those of C. parvum control strain
3 Results and discussion
Development of methods
PCR
The sensitivity of our PCR method for detection of cysts and oocysts
from various matrices was tested by seeded samples. The sensitivity level
for raw water samples and sewage sludge was Giardia (GDH primers) was 125
cysts/sample and Cryptosporidium oocycts also 125/sample. The sensitivity
was increased by hybridisation with specific probes prepared from PCr products
of the respective control strains (Rimhanen-Finne et al. 2001, 2002). Examples
of sensitivity comparisons are shown in Table 1. IC-IF microscopy and IC-PCR
detected same numbers of positive samples for giardia, but microspopy detected
more positive samples for cryptosporidia.
IF-Microscopy
Microcopy was shown to a more sensitive method than PCR because it
detected 1 cyct or 1 oocyst present in the concentrated sample. Microcopy
was shown a necessary method in the studies and it was a complementary
method for PCR. In IF microcopy, the typical morphology is seen, even species
differentiation is not possible. Several Cryptosporidium spp. have similar
size and the immunostaining method did not differentiate between species.
Cry9/cry15 primers are more specific because they will amplify strongly
only DNA of C. parvum, a species pathogenic for humans. IF-microscopy does
not differentiate various Giardia species and, similarly GDH primers used
in our studies are general primers for Giardia spp. Disadvantage of both
PCR and microscopy technique is that they do not distinguish viable or
nonviable cysts or oocycsts.
Concentration
Filtration technique is commonly used for concentration of the samples.
Our results showed that Finnish river raw waters are especially difficult
to filtrate because they have high turbidity values (clay, humic material),
FTU up to140 per litre. Thus even concentration of a10 litre sample needed
two filters and sometimes Envirochek was able to filtrate only 6 litre.
Mussel concentration
We tested a biological system, Finnish river mussels (Unio tumidus
), for concentration of cysts and oocysts. The results are shown in Table
1. These results indicate that mussels efficiently concentrated,
especially Giardia cysts from raw water. Oysters and mussels have been
used also in some other studies for concentration and shown to be efficient
in concetration or biological indicator for contamination of water with
cysts or oocysts. Similarly it v´can be used simultaneously for concentration
of viruses from water samples (see C.H. von Bonsdoff)
Table 1. Comparison of IC-PCR, immunomagnetic capture-imunofluorescence
microscopy
(IC-IF) and using mussels for concentration and IF microcopy for identification
in the detection of Giardia cycts and Cryptosporidium oocysts from raw
water samples.
| Test method | Giardia (% positive) | Cryptosporidium (% positive) |
| IC-PCR (10 liter samples) | 5/20 (25%) | 5/20 (25%) |
| IC-IF (10 liter samples) | 4/20 (20%) | 7/20 (35%) |
| Mussel-IF* | 12/20 (60%) | 4/20 (20%) |
Surface water samples
In the first phase (1999 – 2000), the samples (105) were studied by
IF microscopy after concentration by the method of Vesey et al. (1993)
or Envirochek and only four positive samples for Giardia and Cryptosporidium
were found. When USEPA 1623 method and a commercial immunocapture kit came
available we changed our concentration and purification techniques and
the raw water samples have been treated with that procedure since summer
2000. The samples were collected from 22 rivers and lakes, 5 times 2000
- 2001 and results are shown in Table 2. Some sampling areas were never
contaminated and sampling areas where positive samples were found had some
possible contamination source, such as sewage plant or agricultural area.
Most positive samples were in August and October 2001. If this phenomenon
is a permanent needs further studies with the present methods. Comparison
of different sampling periods is difficult because our techniques have
developed during the study period. For example, in testing August- October
2001 samples, an increased number of 15 freeze-thaw cycles before PCR was
used according to the recommendations of Nicol et al. (IWA, Berlin 2001).
Our results are similar to those reported from Norway and Sweden where
47/147 (35%) or 19/50 (38%) of source water samples were positive, respectively.
Table 2. Giardia and Crytosporidium positive samples collected from 22 rivers and lakes from south-southwestern Finland and tested by PCR.
| Sampling time | Cryptosporidium parvum
Positive/Total number of samples (% positive) |
Giardia
Positive/Total number of samples (% positive) |
Total number of positive watersheds:
Crytosporidium + Giardia |
| October 2000 | 3/22 (13.6%) | 1/22 (4.5%) | 4/22 (18%) |
| February 2001 | 2/30 (6%) | 0/30 (0%) | 2/22 (10%) |
| May 2001 | 1/30 (0.3%) | 0/30 (0%) | 1/22 (5%) |
| August 2001 | 9/30 (30%) | 8/30 (26%) | 16/22 (72%) |
| October 2001 | 13/30 (43%) | 12/30 (40 %) | 8/22 (36%) |
| Total 28/142 (19.7%) | Total 21/142 (14.8%) |
Envirochek samples were collected from 6 water plants during October – December 2000. Of 25 samples, cryptosporidia were detected from two source water samples and giardia were present in none of the samples. The studies continued September – December 2002 at two water plants. At the other plant 2/8 samples were positive either for giardia or Cryptosporidum, and one sample (1/6) from the other plant contained giardia. The samples of river water taken tens of kilometres far away from these drinking water plants had both giardia and cryptosporidia at several occasions during the same period (Table 2). Before the treatment the raw water is stored in reservoirs which may support sedimentation of cysts and oocysts and explain the low frequencies of positive samples detected from raw water at the plant even the volumes studied with Envirochek were higher than those of grab samples (10 l) taken from rivers. These studies showed first time that Finnish watersheds are contaminated by giardia and cryptosporidium. Water from the study area is used as source water for drinking production at several plants. No risk assessment for safety of drinking water for giardia and cryptosporidia has been performed but due to high level of humic material in Finnish rivers the purification systems need full-scale coagulation by Al or Fe and sand filtration. These stages have been shown to be most important in the removal of cysts and oocysts from raw water. Desinfection chemical at most of the surface water plants is chlorine, which is inefficient to destoy of cysts and oocysts. On the other hand, recovery rates of all presently used water methods are low, from a few per cent up to 60 – 70% depending on quality of raw water (Glancy et al. 1999, EU Commision report EC Ref CT 97-2149, 1999). This means that the actual level of positive samples in our studies may be higher because the percentages presented in all Tables indicate the number of positive samples detected.
Follow-up studies of a watershed
A river system was studied from September to October 2001 by taking
samples from 4 municipal sewage treatment plants (effluent, sludge and
influent), raw water samples (10 l) and mussel concentrated samples a few
kilometres lower from the plants, and Envirochek samples from raw water
from the inlet at the drinking water production plant which used river
water as source water.
Table 3. Giardia and cryptosporidia in samples from Vantaa river taken at sewage plants, raw water and drinking water plant
| Sampling place | Sept | Oct I | Oct II | Nov I | Nov II |
| Sewage plant A
River, 5 km lower |
-/-* |
+/+
-/+ |
+/-
+/- |
+/-
+/+ |
+/-
+/- |
| Sewage plant B
River, 2 km lowe |
-/- |
+/-
+/+ |
+/-
+/+ |
+/-
-/- |
+/-
+/- |
| Sewage plant C
River, 1,5 km lower |
-/- |
+/-
+/- |
+/-
+/- |
+/+
+/+ |
ND
-/- |
| Sewage plant D
River 0,35 km lower Drinking water plant |
-/- -/- |
+/-
+/- +/+ |
+/-
+/- ND |
+/-
+/+ -/+ |
+/-
+/- +/- |
These studies (Table 3) showed that a river system, which is a receiving system of influent from several municipal wastewater treatment plants is contaminated by giardia cysts and cryptospordidium oocysts. The concentrations of cysts and oocycts were counted by IF microscopy and they were usually low in river water, 2 to 16/10 l. This level is same as seen in a Norwegian study (Rapport 2000). A great variation has been seen in the numbers of cysts and oocysts per litre (1- appr. 600) and also the the way how these reselts have been obtained is variable. Some studies have counted cysts/oocysts from partial sample in microcope and translate this in to concentration per 100 l, for example.The plant which, was using the river water as source for drinking water has an extremely advanced water purification system which also most probably removes cysts and oocysts.
Sewage sludge samples
A total of 108 raw sludge samples were studied from 24 municipal sewage
plants during spring 2000 and spring 2001. Giardia and Cryptosporidium
was detected in raw sludge by PCR in 8/12 plants in 2000 and in 20/24 plants
in 2001. These 24 plants receive and treat wastewater of approximately
1.8 – 2 million people. Thus the sampling in 2001 was representavite the
whole counry (the whole population 5.2 million).
Genotyping
Genotyping has been now applied for samples positive for C. parvum.
All samples tested, either raw water samples or sewage sludge samples,
have been of genotype 2. A limited number of samples positive for Giardia
have shown to belong to the assemblage B (Belgian type) which is common
e.g. in humans in the Netherlands. This type has a zoonotic
transmission capacity as well. Genotyping studies are still in progress.
Animal faecal samples
Analysis of animal faecal samples is in progress.