Cryptosporidium Cryptosporidium parvum processing Géraldine L.M.C. Duhain

Cryptosporidium Cryptosporidium parvum processing Géraldine L.M.C.  Duhain
Occurrence of Cryptosporidium spp. in South African irrigation
waters and survival of Cryptosporidium parvum during vegetable
processing
By
Géraldine L.M.C. Duhain
Submitted in partial fulfillment of the requirements for the degree
Master of Science
Food Science
In the
Department of Food Science
Faculty of Natural and Agricultural Sciences
University of Pretoria
Pretoria
June 2011
i
© University of Pretoria
DECLARATION
I declare that the dissertation herewith submitted for the degree MSc Food Science at the
University of Pretoria has not previously been submitted by me for a degree at any other
university or institution of higher education.
Géraldine L.M.C. Duhain
ii
ACKNOWLEDGEMENTS
This study was part of an ongoing solicited research project (K5/1773 & K5/1875) funded by the
Water Research Commission, co-funded with the Department of Agriculture.
I wish to express my sincere gratitude and appreciation to the following people and institutions
for their contribution towards the successful completion of this study:
Prof. Elna M. Buys, as study leader, for her mentorship, tremendous support and constant
encouragement throughout this study
Prof. Amanda Minnaar, as co-supervisor, for her advice and constructive criticisms
Lecturers of the Department of Food Science, for their invaluable advice
Dr. Walda van Zyl for her assistance with regards to the PCR analysis
Mrs Riana Cockeran for her assistance with the flow cytometry analysis
The National Research Foundation and the University of Pretoria for their financial assistance
And finally, all my friends and family members for their moral support and companionship
during the completion of this work
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DEDICATION
I dedicate this dissertation to my parents, Françoise and Pierre, and to my siblings, Louise,
Guillaume and Julien.
iv
ABSTRACT
OCCURRENCE OF CRYPTOSPORIDIUM SPP. IN SOUTH AFRICAN
IRRIGATION WATERS AND SURVIVAL OF CRYPTOSPORIDIUM
PARVUM DURING VEGETABLE PROCESSING
By Géraldine Duhain
Supervisor:
Prof. Elna M. Buys
Co-supervisor:
Prof. Amanda Minnaar
Department:
Food Science
Degree:
MSc Food Science
Surface waters used for irrigation purposes in South Africa have been found to be of poor
microbiological quality and to be contaminated with human pathogens. These pathogens can be
transferred from contaminated water onto fresh produce and potentially cause human infections.
Cryptosporidium and Giardia are waterborne parasitic protozoa that have been found in surface
waters worldwide. They can cause morbidity in infected individuals and be lethal when infecting
people with compromised immunity. This study was divided into two phases.
The first phase was a field survey aimed at determining the incidence of human pathogens
Cryptosporidium, Giardia and Salmonella spp. in rivers used for irrigation purposes in South
Africa as well as on vegetables irrigated with these rivers. The rivers selected were from three
different provinces of South Africa. The relationship between faecal indicators and the presence
of Cryptosporidium oocysts and Giardia cysts was also investigated. Out of the 30 water
samples analysed, 43% were positive for Cryptosporidium oocysts, 23% tested positive for
Giardia cysts and 27% were positive for Salmonella spp. However, no Cryptosporidium oocysts
or Giardia cysts were found on the vegetables analysed. No significant differences in the
v
prevalence of Cryptosporidium and Giardia and indicator parameters were observed between the
three rivers. Using a logistic regression model, no significant correlations were observed between
the incidence of faecal indicators and the presence of Cryptosporidium oocysts and Giardia
cysts.
In the second phase of the study, the individual and combined effects of chlorine, blanching,
blast freezing and microwave heating on Cryptosporidium parvum oocysts inoculated on green
peppers were investigated. The viability of the oocysts after treatments was determined using the
vital dye propidium iodide. Stained oocysts were counted with a flow cytometer. Chlorine
treatments did not significantly affect the viability of the oocysts. Blast freezing affected the
viability of 20% of the oocysts. Both microwave heating and blanching affected 93% of oocysts.
Combined treatments with chlorine and blast freezing did not affect the viability of the oocysts
significantly compared to the control. Combined treatment with chlorine and microwave heating
was significantly more effective than microwave heating alone and affected 98.1 % of the
oocysts. The data indicate that C. parvum oocysts are sensitive to heat and, to some extent, to
freezing temperature but are resistant to chlorine.
The results of the survey show the presence of Cryptosporidium and Giardia in irrigation waters
and thus a possible health risk associated with the consumption of raw vegetables as those can
become contaminated via the irrigation water. The results of the challenge tests indicate that C.
parvum oocysts on vegetables are inactivated by blanching and microwave heating but survive
blast freezing and exposure to chlorine. Boiling and microwave heating of vegetables should be
sufficient to kill C. parvum. On the other hand, ready-to-eat vegetables could be at risk of
carrying live C. parvum oocysts as the use of chlorine in washing bath is not expected to
inactivate C. parvum oocysts present on vegetables.
vi
TABLE OF CONTENTS
_____________________________________________________________________________________
LIST OF FIGURES .................................................................................................................................. iv
LIST OF TABLES .................................................................................................................................... vi
CHAPTER 1: INTRODUCTION AND PROBLEM STATEMENT ............. Error! Bookmark not
defined.
CHAPTER 2: LITERATURE REVIEW ............................................. Error! Bookmark not defined.
2.1
WATER SITUATION IN SOUTH AFRICA ......................... Error! Bookmark not defined.
2.1.1 Gauteng province ................................................ Error! Bookmark not defined.
2.1.2 North west province................................................ Error! Bookmark not defined.
2.1.3 Mpumalanga province ............................................ Error! Bookmark not defined.
2.2
WATER USE IN IRRIGATION............................................. Error! Bookmark not defined.
2.3
SOUTH AFRICAN FRUIT AND VEGETABLE INDUSTRY ............ Error! Bookmark not
defined.
2.4
WATERBORNE PROTOZOA PATHOGENS ...................... Error! Bookmark not defined.
2.4.1 Cryptosporidium ..................................................... Error! Bookmark not defined.
2.4.2 Giardia.................................................................... Error! Bookmark not defined.
2.5
WATER CONTAMINATION WITH CRYPTOSPORIDIUM AND GIARDIA..............Error!
Bookmark not defined.
2.6
FOODBORNE OUTBREAKS OF CRYPTOSPORIDIOSIS AND GIARDIASIS ........Error!
Bookmark not defined.
2.7
INDICATOR ORGANISMS .................................................. Error! Bookmark not defined.
2.8
MICROBIAL SOURCE TRACKING .................................... Error! Bookmark not defined.
2.9
SALMONELLA SEROTYPING ............................................ Error! Bookmark not defined.
2.10
EFFECT OF VEGETABLE PROCESSING ON CRYPTOSPORIDIUM OOCYSTS ....Error!
Bookmark not defined.
2.10.1 Effect of chlorine ................................................... Error! Bookmark not defined.
2.10.2 Effect of blanching................................................. Error! Bookmark not defined.
2.10.3 Effect of freezing ................................................................................................... 27
2.10.4 Effect of microwave heating .................................. Error! Bookmark not defined.
2.11
CONCLUSIONS ..................................................................... Error! Bookmark not defined.
2.12
HYPOTHESES ....................................................................... Error! Bookmark not defined.
2.13
OBJECTIVES ......................................................................... Error! Bookmark not defined.
i
CHAPTER 3: RESEARCH ................................................................... Error! Bookmark not defined.
3.1 INCIDENCE OF CRYPTOSPORIDIUM, GIARDIA AND INDICATOR ORGANISMS IN
SOUTH AFRICAN RIVERS USED FOR IRRIGATION OF FRESH PRODUCE .Error! Bookmark
not defined.
3.1.1
Introduction........................................................................... Error! Bookmark not defined.
3.1.2
Materials and methods .......................................................... Error! Bookmark not defined.
3.1.2.1 Sampling sites ....................................................... Error! Bookmark not defined.
3.1.2.2 Water sampling ..................................................... Error! Bookmark not defined.
3.1.2.3 Fresh produce sampling ........................................ Error! Bookmark not defined.
3.1.2.4 Determination of physicochemical parameters ..... Error! Bookmark not defined.
3.1.2.5 Isolation of Cryptosporidium oocysts and Giardia cysts..... Error! Bookmark not
defined.
3.1.2.6 Nested polymerase chain reaction (PCR) ……………………………………….39
3.1.2.7 Isolation of bacterial pathogens ............................ Error! Bookmark not defined.
3.1.2.8 Fresh produce analysis .......................................... Error! Bookmark not defined.
3.1.2.9 Statistical analysis................................................. Error! Bookmark not defined.
3.1.3
Results................................................................................... Error! Bookmark not defined.
3.1.3.1 Incidence of indicator organisms and characterization of protozoa pathogens in
the Klip, Moses and Skeerpoort rivers............................ Error! Bookmark not defined.
3.1.3.2 Incidence of indicator organisms and characterization of protozoa pathogens in
broccoli and letuce ............................................................ Error! Bookmark not defined.
3.1.3.3 Link between indicator organisms and the presence of Cryptosporidium and
Giardia in the three rivers................................................. Error! Bookmark not defined.
3.1.3.4 Link between indicator organisms and the presence of Cryptosporidium and
Giardia in the three rivers................................................. Error! Bookmark not defined.
3.1.4
Discussion ............................................................................. Error! Bookmark not defined.
3.1.5
Conclusions........................................................................... Error! Bookmark not defined.
3.2 EFFECT OF CHLORINE, BLANCHING, FREEZING AND MICROWAVE HEATING ON
CRYPTOSPORIDIUM PARVUM VIABILITY.................................... Error! Bookmark not defined.
3.2.1
Introduction ........................................................................... Error! Bookmark not defined.
3.2.2
Materials and methods .......................................................... Error! Bookmark not defined.
3.2.2.1 Experimental design.............................................. Error! Bookmark not defined.
3.2.2.2 C. parvum oocysts inoculation onto green pepper Error! Bookmark not defined.
ii
3.2.2.3 Effect of chlorine on the survival of C. parvum oocysts inoculated on green
peppers ……………………………………………………………………………….. 59
3.2.2.4 Effect of blanching on the survival of C. parvum oocysts inoculated on green
peppers…………………………………………………………………………………..Er
ror! Bookmark not defined.
3.2.2.5 Effect of blast freezing on the survival of C. parvum oocysts inoculated on green
peppers…………………………………………………………………………………..Er
ror! Bookmark not defined.
3.2.2.6 Effect of microwave heating on the survival of C. parvum oocysts inoculated on
green peppers ………………………………………………………………………….. 60
3.2.2.7 Hurdle effect of combined treatments on the survival of C. parvum oocysts
inoculated on green pepper ............................................... Error! Bookmark not defined.
3.2.2.8 Control .................................................................. Error! Bookmark not defined.
3.2.2.9 Recovery of C. parvum oocysts after treatments .. Error! Bookmark not defined.
3.2.2.10 Staining ............................................................... Error! Bookmark not defined.
3.2.2.11 Flow cytometry analysis ..................................... Error! Bookmark not defined.
3.2.2.12 Statistical analysis............................................... Error! Bookmark not defined.
3.2.3 Results ....................................................................................... Error! Bookmark not defined.
3.2.3.1 Effects of chlorine concentration on Cryptosporidium parvum viability ......Error!
Bookmark not defined.
3.2.3.2 Effects of individual treatments on the viability of Cryptosporidium parvum
oocysts inoculated on green pepper .................................. Error! Bookmark not defined.
3.2.3.3 Hurdle effect of combined chlorination and blast freezing treatment on the
viability of Cryptosporidium parvum oocysts inoculated on green pepper……………...69
3.2.3.4 Hurdle effect of combined chlorination and microwave heating on the viability of
Cryptosporidium parvum oocysts inoculated on green pepper........ Error! Bookmark not
defined.
3.2.4
Discussion ............................................................................. Error! Bookmark not defined.
3.2.5
Conclusions........................................................................... Error! Bookmark not defined.
CHAPTER 4: GENERAL DISCUSSION ........................................... Error! Bookmark not defined.
4.1 CRITITCAL REVIEW OF METHODOLOGY AND EXPERIMENTAL DESIGN.............Error!
Bookmark not defined.
iii
4.2 CONSEQUENCES OF THE PRESENCE OF CRYPTOSPORIDIUM IN IRRIGATION WATER
AND THE EFFECTS OF TREATMENTS ON C. PARVUM VIABILITY ……. ....Error! Bookmark
not defined.
CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS ... Error! Bookmark not defined.
CHAPTER 6: REFERENCES .............................................................. Error! Bookmark not defined.
iv
LIST OF FIGURES
______________________________________________________________________________
Figure 1:
Map showing major rivers of the Gauteng Province (a), the North West Province
(b) and the Mpumalanga province (c) (DWA, 2010) .......... Error! Bookmark not
defined.
Figure 2:
Map of South Africa showing the sampling sites (Newsbank Reference Maps,
2010) ....................................................................Error! Bookmark not defined.
Figure 3:
Aerobic colony count (ACC), faecal coliforms counts, E. coli counts and
incidence of Salmonella spp., Cryptosporidium and Giardia in the Klip (A),
Skeerpoort (B) and Moses rivers (C) for the 10 sampling intervals between June
2009 and April 2010 ..............................................Error! Bookmark not defined.
Figure 1:
Amplified Cryptosporidium DNA on agarose gel. Lane 1-6: slides from water
samples, 7-8 positive control slides, 9 PCR positive control, 10 PCR negative
control, lanes M:100 bp DNA ladder ……………………………………………46
Figure 5:
Effects of different levels of chlorine on the survival of C. parvum oocysts
inoculated on green pepper (n=6). .........................Error! Bookmark not defined.
Figure 6:
Percentages C. parvum oocysts positive for PI fluorescence after treatment with
100 ppm chlorine. ..................................................Error! Bookmark not defined.
Figure 7:
Effect of chlorination, blanching, microwave heating and blast freezing treatments
on the survival of C. parvum oocysts inoculated on green pepper (n=6). ..... Error!
Bookmark not defined.
Figure 8:
Percentages C. parvum oocysts positive for PI fluorescence after no treatment
(Control) (A), after treatment with 200ppm chlorine (B), after blanching (C), after
blast freezing (D) and after microwave treatment (E). ........ Error! Bookmark not
defined.
Figure 9:
(1) Analysis with flow cytometer of C. parvum on bivariate dotplots of size (FS)
versus green fluorescence (PTM2). The gate (A) was defined to include all FITC
fluorescent cells. (2) Percentage Cryptosporidium oocysts positive for PI
fluorescence after blanching. .................................Error! Bookmark not defined.
v
Figure 10:
Effect of individual chlorination and blast freezing treatments or combination of
both treatments on the survival of C. parvum oocysts inoculated on green pepper
(n=6).......................................................................Error! Bookmark not defined.
Figure 11:
Percentage C. parvum oocysts positive for PI fluorescence after combined
treatments with 200 ppm chlorine and blast freezing. ......... Error! Bookmark not
defined.
Figure 12:
Effects of individual chlorination and microwave heating treatments or
combination of both treatments on the survival C. parvum oocysts inoculated on
green pepper (n=6).................................................Error! Bookmark not defined.
Figure 13:
Percentage C. parvum oocysts positive for PI fluorescence after combined
treatments with 200 ppm chlorine and microwave heating. Error! Bookmark not
defined.
Figure 14:
Model indicating the relationship between initial level of hazard on raw product,
effect of blast freezing, and food safety risk of final product .....Error! Bookmark
not defined.
Figure 15:
Model indicating the relationship between initial level of hazard on raw product,
effect of microwave heating or blanching, and food safety risk of final product
................................................................................Error! Bookmark not defined.
Figure 16:
Model indicating the relationship between initial level of hazard on the raw
product, effect of chlorine, microwave heating and blast freezing, and food safety
risk of final product................................................Error! Bookmark not defined.
vi
LIST OF TABLES
______________________________________________________________________________
Table 1:
Reported foodborne outbreak of cryptosporidiosis and giardiasis ................ Error!
Bookmark not defined.
Table 2:
Indicator parameters and percentage of samples positive for pathogens in the
vegetable samples analysed ...................................Error! Bookmark not defined.
Table 3:
Indicator parameters and percentage of water samples (n=30) positive for
pathogens for the samples collected between June 2009 and May 2010 from the
Klip, Moses and Skeerpoort rivers.........................Error! Bookmark not defined.
Table 4:
Percentage of water samples from the Klip, Moses and Skeerpoort rivers (n=30)
positive for Salmonella spp., Cryptosporidium and Giardia at various levels of
faecal coliforms in samples....................................Error! Bookmark not defined.
Table 5:
Percentage of water samples from the Klip, Moses and Skeerpoort rivers (n=30)
positive for Salmonella spp., Cryptosporidium and Giardia at various levels of E.
coli in samples........................................................Error! Bookmark not defined.
Table 6:
Logistic regression analysis of relationship between indicator parameters and
presence of Cryptosporidium and Giardia p-valueError! Bookmark not defined.
Table 7:
t-test analysis of % inactivated oocysts after 100 ppm chlorine and 200 ppm
chlorine treatments compared to the control..........Error! Bookmark not defined.
Table 8:
t-test analysis of % inactivated oocysts after chlorine, blanching, blast freezing
and microwave heating treatments compared to the control group ............... Error!
Bookmark not defined.
Table 9:
t-test analysis of % inactivated oocysts after individual and combined chlorine
and blast freezing treatments compared to the control group .....Error! Bookmark
not defined.
Table 10:
t-test analysis of % inactivated oocysts after individual and combined chlorine
and microwave heating treatments compared to the control group ............... Error!
Bookmark not defined.
vii
CHAPTER 1: INTRODUCTION AND PROBLEM
STATEMENT
________________________________________________________________________
According to Beuchat (2002), foodborne infection outbreaks associated with the
consumption of raw vegetables have been increasing world-wide. One of the reasons for
this increase is the changes in consumers’ dietary habits. People are becoming more
concerned about their health and have increased their consumption of fresh and
minimally processed fruits and vegetables. They demand ready-to-eat fresh products, free
from chemical preservatives (Zink, 2009). Also, in order to increase the vitamin intake of
populations in developing countries, nutritionists recommend these populations grow
their own vegetable gardens and consume their own vegetables (Zachariah, Teck,
Buhendwa, Labana, Chinji, Humblet and Harries, 2006). Microbiological results from
irrigation water and vegetables in South Africa have however indicated that the downside of consuming more fresh vegetables is the possible increased exposure to pathogens
and thus increased risks of infection, especially if the vegetables are consumed in the raw
state (Britz, Sigge and Barnes, 2008).
Because water resources in South Africa are limited, most farmers rely on untreated
surface water for irrigation of their vegetable crops. However, surface water can be of
poor microbiological quality and could contaminate the vegetables with pathogenic
organisms since contaminated water is deposited onto the surface of the crops during
irrigation (Robertson and Gjerde, 2001a). High levels of Escherichia coli, indicator of
faecal pollution as well as bacterial pathogens Listeria monocytogenes, Staphylococcus
aureus and Salmonella spp. have been recovered in rivers from Western Cape,
Mpumalanga, Gauteng and North West Province (Britz et al., 2008). Moreover, diarrhea
outbreaks of waterborne sources have been reported in Delmas and Standerton therefore
showing the impact of water pollution on the population’s health in South Africa (NICD,
2007; NICD 2008).
Apart from the pathogens already mentioned, pathogenic protozoa can also be found in
untreated surface water. Cryptosporidium and Giardia are two of the most common and
resistant human waterborne parasitic protozoa (Chaidez, Soto, Gortares and Mena, 2005).
They both have been associated with severe outbreaks of diarrhea in humans. They cause
morbidity in healthy individuals and can be lethal in susceptible population.
Cryptosporidium and Giardia are regularly isolated from raw water in the Gauteng area
(Monique Grundlingh, Rand Water, personal communication, 2008). In a survey done by
Kfir, Hilner, du Preez and Bateman (1995), Cryptosporidium and Giardia were found in
South African surface water, treated effluent and drinking water. Because of the high cost
and low recovery rate of the isolation methods for Cryptosporidium and Giardia, it is
important to assess the correlation between the presence of faecal indicator organisms
such as E. coli, coliforms and incidence of Cryptosporidium and Giardia. Few studies
have been done in South Africa to determine the simultaneous incidence of
Cryptosporidium and Giardia and faecal indicators. Therefore one of the objectives of
this study is to determine whether the presence of faecal indicators can be linked to the
presence of Cryptosporidium oocysts and Giardia cysts in irrigation water and on
vegetables in South Africa.
Most reported cryptosporidiosis and giardiasis outbreaks have been associated with
drinking water. However, Cryptosporidium oocysts have been found on the surface of
raw vegetables in both developing and developed countries (Robertson and Gjerde,
2001b). The transmissive stage of these parasites can survive for long periods of time
especially in moist environment such as those surrounding fresh produce. Furthermore
Cryptosporidium oocysts and Giardia cysts are resistant to chlorine at the concentration
and contact times usually used for washing vegetables and thus might not be eliminated
by washing (Chaidez et al., 2005). The cysts and oocysts could therefore survive on both
washed and unwashed vegetables and cause foodborne infections. Also, the presence of
the pathogens on export produce could lead to serious economical losses if fruits are
rejected and exportation suspended (Britz, Sigge and Barnes, 2008).
Apart from being washed, vegetables can also be blanched and frozen before being
retailed. Once bought, vegetables can also be cooked either in hot water or in a
microwave before being consumed. These treatments might affect the viability of
Cryptosporidium oocysts. Little is known about the hurdle effect of combined treatments
on the survival of oocysts on vegetables. This study will thus investigate the individual as
well as combined effects of chlorine, blanching, freezing and microwaving on
Cryptosporidium oocysts structure when inoculated on fresh produce.
CHAPTER 2: LITERATURE REVIEW
________________________________________________________________________
______
2.1
WATER SITUATION IN SOUTH AFRICA
Water is indispensable to life and is essential for food production, hygiene, sanitation and
for economic development. However, water resources in South Africa are scarce and
limited since a large part of South Africa is located in a semi-arid part of the world. The
average annual rainfall for the country is 495 mm, and ranges from 100 mm/year in the
western desert to about 1200 mm/year in the eastern part of the country (FAO, 2005).
Water resources in South Africa are made up of surface water and ground water. Surface
water resources include rivers, dams, pans, wetlands and dolomite eyes fed by
underground water sources. The river network in South Africa can be divided into four
major systems, namely the Orange river system which includes the Caledon and the Vaal
rivers, the Limpopo system in the North which includes the Olifants and Crocodile rivers,
the Tugela river and the Olifants river system in the south-western part of the country
(FAO, 2005). Groundwater is the term used to describe underground water resources,
which flow between soil pore spaces and within underground rock formations (NWPEO,
2008).
Renewable, water is a finite resource, which requires careful management and protection.
It is important for water to be of acceptable quality for human and other uses. Water is
highly susceptible to pollution and continued deterioration of water quality in some parts
of South Africa has been reported (DACE, 2004). Bacteriological contamination of
water sources is wide spread in the country and can lead to waterborne disease outbreaks.
One of the major sources of microbiological pollutions are the many informal settlements
with no or poorly maintained sanitation facilities that have been established near rivers.
Runoff waters from these areas carry high levels of feacal matter of human origin that
can possibly contain human pathogens (Britz, 2007). Increased urban development has
caused an increased pollution load. This together with lack of adequate water treatment
plants and sewerage work maintenance has led to an increase in sewage effluent being
released into surface water in recent years (Groenewald, 2010a, 2010b). Pollutants from
mining, pesticides used in agriculture, leach from waste disposal facilities and
uncontrolled dumping near water sources are contributing to the increased pollution of
our surface waters (Groenewald, 2009).
The demand on South Africa’s water resource is increasing due to urban and agricultural
development. Climate change is expected to increase even further the variability and
intensity of rainfall putting even more pressure on the already scarce water resources.
River flow in some parts of the country is expected to drop by up to 58% by the end of
the century (De Wit and Stankiewicz, 2006). Already 11 of the 19 water management
areas in South Africa are facing a water deficit (based on 2000 figures) (MSER, 2003).
The water situation in the provinces included in this study, namely the Gauteng, North
West Province and Mpumalanga, will now be discussed in more details.
2.1.1
GAUTENG PROVINCE
Gauteng Province includes three water management areas namely the Crocodile WestMarico, Upper Vaal and Olifants river (Figure 1a). Due to the high demand for water, the
province can not only rely on natural surface runoff and ground water but must import
raw water from outside the province (DACE, 2004)
a)
b)
c)
Figure 1: Map showing the major rivers of the Gauteng province (a), the North
West province (b) and the Mpumalanga province (c) (DWA, 2010)
Demand for water in Gauteng is increasing due to the increasing population density and
economic development. The mineral industry and growing agricultural activities put an
extra strain on the water resources. Furthermore, increased runoff from increasing
urbanisation is causing a loss to the ground water recharge potential (DACE, 2004).
Aquifer de-watering and re-watering caused by mining activities is a big problem in
Gauteng. This causes water to migrate from elevated regions to lower lying areas thus
leaving some areas without water (DACE, 2004).
The microbiological quality of the Klip river was monitored in this study. Agriculture is
one of the main users of water from the Klip river catchments and it was estimated that 4
400 ha of land could be irrigated in the catchments (Kotze, 2002). Treated sewage
effluent from Johannesburg’s southern wastewater treatment work are used to irrigate
crops in the upper Klip river while sewage sludge from the East Rand Water Care Works
are used for irrigation in the area of Klip river and Rietspruit (Kotze, 2002). Maize,
fodder crops and vegetable such as carrots, spinach, cabbage, onions, potatoes and salad
greens are the main irrigated crops grown in the Klip river catchments area (Kotze,
2002).
The quality of surface water in Gauteng has been found to be poor due to chemical
pollution from mines and microbiological pollution from informal settlements and faulty
sewage treatment works (DACE, 2004; Groenewald, 2010a). The sulphate/chloride
ration, which is used to indicate the effect of mining on water salinity, has been found to
be elevated in the Blesbokspruit, Klip river and Wonderfonteinspruit showing the
polluting effect of mining in those areas. High levels of total dissolved solids were also
found in those waters indicating discharge from industrial and mining activity. The levels
of faecal coliforms in the Klip river and Blesbokspruit have been found to be elevated
since 2000. This shows pollution of these rivers with faecal material that could most
likely be carrying human pathogens (DACE, 2004).
In 2003, the ecological status of rivers in Gauteng was described as poor to fair. This
means that the natural functioning of the rivers had been disrupted and the rivers
ecosystem extensively used (River Health Program, 2003). The fish population, which is
used as a good indication of the long term influences of pollution on a river, has been
described as poor in the Upper Klip river, Natalspruit river, Lower Klip river,
Suikerbosrand river, Rietspruit river and Blesbokspruit river (DACE, 2004).
Most of the major dams in Gauteng suffer from high levels of eutrophication which is
caused by enrichment of water with plant nutrients, especially nitrogen and phosphorus
compounds. Hyper eutrophication can cause serious health effects. The process of
eutrophication takes thousands of years to occur naturally but in this case unnatural
eutrophication was a direct result of agricultural and urban runoff, municipal and
industrial wastewater effluents, and septic tanks that all contribute organic nutrients
(DACE, 2004).
2.1.2
NORTH WEST PROVINCE
The four management areas in North West are the lower Vaal, middle Vaal, upper Vaal
and Crocodile Marico river (Figure 1b) (NWDACE, 2008).
The ground water in North West is of particular importance as 80% of the water used in
rural and agricultural activities comes from ground water (NWDACE, 2008). Depletion
of the ground water resources is caused by use of water for irrigation, drinking and stock
watering as well as by removal or transfer of water associated with mining activities and
industrial development (NWDACE, 2008). Irrigated agriculture is the biggest user of
water in North West and requires 418 million m3 per annum, most of which come from
ground water. Vegetable crops grown under irrigated conditions in the North West
Province include potatoes, cabbage, peppers, beans and tomatoes (NWPG, 2010).
Ground water in North West was found to be of reasonable quality according to a study
done by Zitholele Consulting (2008). However high nitrate and total dissolved solids
concentrations were found in some regions. High level of sulphate due to acid mine
drainage from gold mines in the far West Rand were also observed (NWDACE, 2008).
The surface water resources in North West province are under pressure due to population
growth, mining, agricultural and industrial water uses (NWDACE, 2008). The surface
water resources are scarce as many surface water systems are non perennial. More
surface water is available in the east of the province than in the west. The main sources
of surface water pollution in the North West province are diffusion of fertilizers run-off,
insecticides and herbicides from agricultural land and storm water runoff from urban
surfaces such as roads as well as diffusion from dense settlements. The rivers in North
West province are also polluted by effluent from mining and industrial activities
(NWDACE, 2008).
High levels of nitrate and nitrites were found in the rivers of the North West province
which is indicative of high nutrient status. This is associated with agriculture and
industrial pollution e.g. discharges of sewage effluent in the rivers (DACE, 2004;
NWDACE, 2008). High levels of nitrite can lead to eutrophication and algal bloom in
dams. Eutrophication impacts negatively on tourism and recreational sport as it leads to
unpleasant odours, kills the fish and causes a potential health risk for the recreational
water users. High faecal coliforms levels have been observed in rivers in North West
province between 2002 and 2007. The faecal coliform counts were above the Department
of Water Affairs and Forestry (DWAF) standard of 2000 faecal coliforms count/100ml
for water for domestic and recreational water. The counts were also above the World
Health Organisation (WHO) standard of 1000 faecal coliform count/100 ml in irrigation
water (NWDACE, 2008; WHO, 1989). Faecal coliforms indicate pollution of the rivers
with faecal matter which indicates possible contamination with human pathogens (Scott,
Rose, Jenkins, Farrah and Lukasik, 2002). Effluents from sewage treatment plants as well
as runoff from poorly sanitised settlements are the probable cause for these high faecal
coliform counts. The North West province has the highest number of informal
settlements of the country as 23% of the household are described as informal (Statistics
South Africa, 2007). Sanitation in these settlements is poor and water runoffs from these
areas contain high levels of faecal matter.
2.1.3
MPUMALANGA PROVINCE
The sources of four of South Africa’s major river systems are found in Mpumalanga,
namely the Olifants river system, the Orange river system (Vaal river), the Inkomati river
system (Crocodile, Sabie, Sand and Komati rivers), and the Pongola river systems
(Figure 1c) (MSER, 2003).
Most of the water available in Mpumalanga comes from surface water (65%). Water
transfers from outside the province provides 19% of the total available water while only
6% of the water comes from ground water (MSER, 2003). Return flow from mining,
industrial, urban sectors and from irrigation account for 10% of the total water available
in Mpumalanga. In Mpumalanga, 46% of the water available is used by farmers to
irrigate their crops (MSER, 2003). However, the demand for irrigation water is unevenly
distributed throughout the province with 28% of water requirement for irrigation coming
from the middle Olifant river area alone in the North East of the Province. This is where
the Loskop dam and Loskop dam irrigation scheme are located (MSER, 2003). Wheat,
vegetables, tobacco, peanuts, cotton and citrus fruit are cultivated and irrigated under the
Loskop dam irrigation scheme in Mpumalanga (Loskop Irrigation Board, 2009).
However, increased pollution levels in the Olifant river have been reported in the media
creating concerns about the safety of fresh produce and the risk of loosing exportation
deals (Groenewald, 2010b).
There has been a general decrease in water quality in Mpumalanga since 1996 as shown
by various quality indicators such as water nutrient level, total dissolved solids, pH and
metal concentration (MSER, 2003). The eutrophication levels in Mpumalanga’s dams
were within acceptable levels in 2002. The Loskop and Witbank dams were described as
mesotrophic while the Middelburg dam was oligotrophic which means that these dams
contain low levels of organic matter. The Grootdraai dam, however, was found to be
eutrophic which means it contains higher levels of nutrients and algae. High levels of
total dissolved solid content have been found in the upper Vaal and Olifants water
management areas (MSER, 2003). These high levels usually indicate discharge from
industrial and mining activities (DACE, 2004). High levels of sulphate and heavy metals
were also observed in the surface waters of the province and these levels exceeded
standard set in the South African drinking water quality guidelines for industrial and
irrigated agricultural use (MSER, 2003).
2.2
WATER USE IN IRRIGATION
Agriculture practices in South Africa are limited by the availability of water. Due to low
rainfall and high incidence of dry spells, farmers in most part of the country are
dependent on irrigation as 65% of the country does not receive sufficient rainfall for
successful rain-fed crop production (FAO, 2005). It is thus not surprising that irrigation is
the main user of water in South Africa accounting for 62% of water withdrawal in the
country (MSER, 2003; FAO, 2005). The main irrigated crops are fodder crops, wheat,
maize, vegetables and pulses. Horticulture is a major user of water for irrigation;
vegetable production alone accounted for 8% of total harvested irrigated cropped area in
South Africa in 2000 (FAO, 2005).
Because clean water resources in South Africa are limited, farmers often have to use
water sources of poor quality to irrigate their crops. Water used for irrigation can be
classified according to their composition and where they originate from (Britz, 2007).
Raw water refers to untreated water from surface water sources such as rivers and dams
or groundwater from wells. Wastewater refers to water that has already been used for
domestic or industrial use. Wastewater can be divided into sullage and black water.
Sullage includes household grey water but excludes water from toilets. Black water refers
to toilet waste. In poor populations living in informal settlements, wastewaters contain
high concentrations of human excreta and thus possibly high levels of human pathogens.
Storm water refers to water generated from runoffs from house roofs, paved areas and
roads during rainfall. Effluents from water treatment plants are returned to the nearest
natural water surface to make a contribution to the river ecology. Storm water and
effluent from water treatment plants contribute to the flow and microbiological quality of
surface waters. This contribution can be beneficial or detrimental depending on the
efficiency of the water treatment plant and the quality of the effluent being discharged in
the rivers (Britz, 2007). Unfortunately the poor maintenance and over loading of water
treatment plants have been reported in South Africa. Dumping of raw sewage and
effluent of poor quality into rivers has thus resulted in contamination of rivers with high
levels of faecal material (Groenewald, 2009).
The use of water of poor microbiological quality to irrigate fresh produce could constitute
a public health hazard as human pathogens can be transferred to crops during irrigation
(Armon, Gold, Brodsky and Oron, 2002). This is of particular importance if water
containing pathogens is used to irrigate crops that will not be cooked prior to
consumption (Tyrrel, 1999).
2.3
SOUTH AFRICAN FRUIT AND VEGETABLE INDUSTRY
In 2003, agriculture contributed 3.8% to the GDP and employed 8% of the labour force in
South Africa (FAO, 2005). South Africa is a net exporter of food products. Maize is
South Africa’s main farm export while other major crops include sugar cane, wheat,
potatoes, groundnut, citrus fruit and grapes (FAO, 2005). According to the National
Department of Agriculture (DOA, 2009), the total quantity of the most important
vegetables sold on fresh produce market increased from 2 046 300 tons in 1997 to 2 115
400 tons in 2008. In 2008, the major contributors of important vegetables sold on fresh
produce markets were potatoes (1853 000 tons), tomatoes, (426 000 tons), onions (375
000 tons), green mielies (323 000 tons) and pumpkins (226 000 tons) (DOA, 2009).
South Africa is the major and leading exporter of fruits and vegetables in Africa. South
Africa holds 31% percent of share in the European Union (EU) market and is now the
largest third-country supplier of fruits and vegetable to the EU. Just behind is Turkey
with 29% share and Morocco with 22% share in the fruit and vegetable EU market (Diop
and Jaffee, 2005). Several countries in Sub Saharan Africa export fruits and vegetables;
however three countries dominate the market. South Africa, Cote d’Ivoire and Kenya
account for 90 % of international export of fruits and vegetables in Sub Saharan Africa
with South Africa as the leading exporter (Diop and Jaffee, 2005). South Africa, together
with Chile and Argentina dominate the exports market for apples, grapes and pears to the
European Union. South Africa is also a major exporter of fruits and vegetables to the
United States (Diop and Jaffee, 2005).
It is clear that fruits and vegetables play a major role in South Africa’s economy. South
African farmers and retailers are concerned over the state of the rivers and the impact it
could have on the quality and safety of their products. Major retailers are worried to loose
exportation deals with Europe due to the presence of pathogens on their fresh produce
(Tempelhoff, 2010). It is thus important to avoid contamination of fresh produce with
pathogens as outbreak of foodborne illnesses as a result of consumption of South African
fresh products would lead to banning export of such produce to overseas countries which
would lead to severe economical losses (Arnade, Calvin and Kuchler, 2009).
2.4
WATERBORNE PROTOZOA PATHOGENS
2.4.1 CRYPTOSPORIDIUM
Cryptosporidium is an obligate, intracellular, eukaryotic protozoon from the phylum
Apicomplexa. The first cases of cryptosporidiosis were reported in 1976 and
cryptosporidiosis infections have now been reported in over 90 countries and six
continents (Fayer, Morgan and Upton, 2000).
Cryptosporidium spp. infects both human and animals and over 152 species of mammals
have been reported to be infected with Cryptosporidium (Fayer et al., 2000).
Cryptosporidium hominis is the specie that causes most human cryptosporidiosis and
Cryptosporidium parvum is typically found in ruminants but can also infect humans
(Dawson, 2005). Other species of Cryptosporidium have also been reported to infect
humans and zoonotic transmission from animal to human is thus possible (MMWR,
2010).
Cryptosporidium has a two-stage life cycle consisting of a reproductive stage and an
environmentally resistant oocyst stage. Oocysts are spore-like survival structures,
resistant to environmental stresses and commonly used disinfectants such as chlorine
(Moriarty, Duffy, McEvoy, Caccio, Sheridan, McDowell and Blair, 2005). The great
resistance of Cryptosporidium oocysts to chemical disinfectants is attributed to their
oocysts walls which are made of a double layer of protein-lipid-carbohydrate matrix
(Templeton, Lancto, Vigdorovich, Liu, London, Hadsall and Abrahamsen, 2004).
Oocysts are spherical in shape, measure between 4 to 6 µm and contain four sporozoites.
Amylopectin is the polysaccharide found in coccidian protozoa and provides energy for
excystation and penetration of host cells (Harris, Adrian and Petry, 2003). Small amounts
of amylopectin are also used during the dormancy stage and Cryptosporidium oocysts
have been found to be unable to infect host once the amylopectin store has been depleted
(Vetterling and Doran, 1969).
Oocysts are the stage transmitted from an infected host to a susceptible host by the faecal
oral route (Fayer et al., 2000). Oocysts can be transmitted from person to person, from
animal to human, through drinking contaminated water or eating contaminated food and
from swimming in contaminated water (Fayer et al., 2000; MMWR, 2010). Once
ingested, oocysts release the reproductive stage: the sporozoites. Sporozoites attach
themselves to the intestinal epithelium and multiply, damaging the mucous membrane of
the intestinal lining and causing diarrhea (Dawson, 2005). New oocysts are then formed
and shed in the faeces of infected host. Cryptosporidium has a low infective dose and the
ingestion of as few as 10 oocysts has been shown to cause infections. The infective dose
varies from species to species but the average infective dose has been calculated to be 87
oocysts (Okhuysen, Chappell, Crabb, Sterling and Dupont, 1999). The incubation period
varies between 3-7 days (Tzipori and Ward, 2002). Cryptosporidium infections are
usually self limiting in healthy individuals but can become chronic and life threatening in
individuals with weakened immune systems (Dawson, 2005; Lane and Lloyd, 2002).
Populations especially at risk of infections are children, malnourished persons, and
individuals with compromised immunity including AIDS patient, transplant recipient,
patients receiving chemotherapy for cancer, institutionalized patients and patients with
immunosuppressive infectious diseases (Fayer et al., 2000). Up to 2002, no drug therapy
had been found effective against Cryptosporidium infections. Treatment for
cryptosporidiosis is mainly symptomatic and supportive (Fayer et al, 2000). In 2002,
Nitazoxanide was the first and only broad spectrum anti-parasitic drug approved for use
in the United State for the treatment of cryptosporidiosis (MMWR, 2010).
2.4.2 GIARDIA
Giardia lamblia is a waterborne, flagellated protozoon from the order Diplomonadida
and was first discovered in 1681 by Antoine Van Leeuwenhoek (Lane and Lloyd, 2002).
The Giardia species infecting humans are Giardia intestinalis and Giardia duodenalis
sometime called Giardia lamblia. Giardia lamblia is endemic throughout the world with
a high incidence in the tropics and subtropics. It infects humans, mammals, reptile and
birds (Lane and Lloyd, 2002). Giardiasis is now recognized as a traveler’s disease
worldwide. It is one of the most common source of intestinal infection in the developed
world as well as a serious cause of infection in developing countries (Stevens, 1982;
Swaminathan, Torresi, Schlagenhauf, Thursky, Wilder-Smith, Connor, Schwartz,
VonSonnenberg, Keystone and O-Brien, 2009).
Giardia lamblia has a two stage life cycle and alternates between trophozoite and cysts.
The trophozoite is the vegetative reproductive stage. It is pear shape and 9.5 to 21 µm
long by 5 to 15 µm wide, has four pairs of flagellae and two nuclei. The trophozoite has a
ventral sucking disk made of microtubule which helps with attachment to the intestinal
mucosal layer of its host (Wolfe, 1992). Cysts are the survival stage and the infectious
form that is found in the environment (Lane and Lloyd, 2002). They are ovoid, measure 8
to 12 µm long by 7 to 10 µm wide and contain four nuclei. Cysts have a hyaline cyst wall
which makes them resistant to chlorination and they can survive for several months in
cold surface water (Lane and Lloyd, 2002).
Infection occurs when cysts are ingested with contaminated food or water. Cysts pass
through the stomach undamaged and excyst once they reach the duodenum where the
alkaline pH is favourable for the growth of the trophozoites. Two trophozoites are
released from each mature cyst. Some trophozoites then attach themselves to the
intestinal villi with the help of the sucking disks while others swim freely in the
duodenum and ileum (Lane and Lloyd, 2002). Trophozoites replicate asexually by binary
fission inside the intestine, damaging the epithelial cells and causing the symptoms of
giardiasis. New cysts are then formed which are discharged in stools of infected hosts and
then transmitted to new host through food or water (Thompson, 2000). Humans are the
main reservoir of Giardia but animals can carry species of Giardia similar to those
infecting humans (Wolfe, 1992). Calves are also a big reservoir of Giardia with a high
incidence of infection worldwide (Olson, Thorlakson, Deselliers, Morck and McAllister,
1997). Gardia duodenalis which includes G. lamblia naturally infects humans, beavers,
coyotes, cattle, cats and dogs (Wolfe, 1992). Species of Giardia originating from animals
have been reported to be infectious to humans as well (Wolfe, 1992). Shedding of high
numbers of cyst in the environment by livestock is thus a public health concern as this
could lead to zoonotic infections (Lane and Lloyd, 2002).
Giardiasis infection occurs by feacal-oral contamination with cysts or indirectly through
consumption of contaminated food or water. Ingestion of 100 or more cysts is required to
ensure infections in humans but as few as 10 cysts have resulted in infections in
volunteers. This is due to the fact that a single ingested cyst will divide and quickly
multiply to infectious levels (Lane and Lloyds, 2002). Travellers often become infected
by drinking contaminated ground or surface water. The incubation time varies between 9
to 15 days and the symptoms of the disease are variable but include nausea, low fever,
watery diarrhea and malabsorption (Wolfe, 1992; Thompson, 2000). The acute stage lasts
between 3 to 4 days and usually clears spontaneously in individuals with healthy immune
system. However, chronic infection may develop and last two or more years or even
become fatal in individuals with compromised immunity (Wolfe, 1992). Yet, around 13%
of infected adults and up to 50% of infected children remain asymptomatic (Wolfe,
1992). Several drugs are available to treat the infection. In South Africa the use of
metronidazole is recommended (National Department of Health, 1998).
2.5 WATER CONTAMINATION WITH CRYPTOSPORIDIUM AND
GIARDIA
Cryptosporidium and Giardia have mainly been associated with waterborne outbreaks.
Cryptosporidium oocysts are ubiquitous in surface water worldwide (WHO, 2008) and
have been found in untreated surface water (Ong, Moorehead, Ross and Isaac-Renton,
1996), ground water (D’Antonio, Winn, Taylor, Gustafson, Current, Rhodes, Gary and
Zajac, 1985), treated drinking water (D’Antonio et al., 1985), recreational water and even
bottled water (Franco and Neto, 2002). Kfir, Hilner, du Preez and Bateman (1995)
investigated the presence of Cryptosporidium and Giardia in surface water in South
Africa. They found 30% of samples tested positive for Giardia cysts while 2.9% of
samples tested positive for both Giardia and Cryptosporidium (Kfir et al., 1995).
The first confirmed documented waterborne outbreak of cryptosporidiosis occurred in
Texas in 1984 and was caused by unfiltered ground water supply (D’Antonio et al.,
1985). The number of reported cases of cryptosporidiosis has been increasing ever since
and the centre of disease control in the United State has reported an increased from 6 479
cryptosporidiosis cases in 2006 to 10 500 cases in 2008 (MMWR, 2010). On the other
hand, giardiasis cases are on a slight decline and 19 140 cases of giardiasis were reported
in the United State during 2008 while
19 238 cases were reported in 2006. More
cases of cryptosporidiosis were reported in summer than in winter. This was mainly
attributed to the increase in recreational water-associated outbreaks due to
Cryptosporidium oocysts’ resistance to chlorine (MMWR, 2010). Recreational waterassociated outbreaks of giardiasis are also well documented and Giardia was found to be
responsible for 3.7% of recreational water-associated gastroenteritis outbreaks in the
United States between 1997 and 2006 (MMWR, 2010).
Humans and animals act as a reservoir of Cryptosporidium and Giardia. In a study
carried out in 1986 in a Durban hospital, South Africa, Cryptosporidium was the second
most common organism detected among children admitted with diarrhoea (Van den
Enden, 1986). In Limpopo, South Africa, a Cryptosporidium incidence of 18% was
observed in school children and hospital patients (Samie, Bessong, Obi, Sevilleja, Stroup,
Houpt and Guerrant, 2006).
It has been estimated that 32% of AIDS patient have
suffered from cryptosporidiosis at some stage in their life (Hunter and Nichols, 2002
according to Moore et al., 2007). Cattle and calves are big reservoirs of the parasites.
High prevalence of Giardia and Cryptosporidium has indeed been reported (Olson et al.,
1997; Lefay, Naciri, Poirier and Chermette, 1999). Furthermore, higher Cryptosporidium
seroprevalence has been observed among dairy farmers compared to other farmers which
suggest zoonotic transmission from cattle to human (Fayer, Dubey and Lindsay, 2004).
Large amount of cysts or oocysts are shed in faeces of infected hosts. As much as 10 10
Cryptosporidium oocysts or 109 Giardia cysts can be released daily from a host during
acute or chronic infection (Tzipori and Ward, 2002). Excretion of infectious
Cryptosporidium oocysts can carry on up to 50 days after the diarrhea has stopped while
shedding of infectious Giardia cysts can carry on for months (Tzipori and Ward, 2002;
MMWR, 2010). In South Africa, water sources located near farms or informal
settlements are thus more likely to become contaminated with Cryptosporidium oocysts
and/or Giardia cysts. Water sources can become contaminated through run off water
from infected fields or poorly sanitized settlements and direct contamination by infected
animal and human faeces is also likely to occur. Rivers downstream of cattle farms have
indeed been found to contain higher levels of Cryptosporidium oocysts and Giardia cysts
than water upstream of the farms (Ong, Ross and Isaac-Renton, 1996).
The many outbreaks associated with drinking water confirm that purification systems can
fail to eliminate Cryptosporidium oocysts and Giardia cysts (Fayer, Morgan and Upton,
2000). Improperly designed conventional water treatment using sand filtration and
chlorine systems may not remove all oocysts or cysts. A typical example of water
contamination is the Milwaukee outbreak in 1993. It was caused by Cryptosporidium
oocysts passing through one of the drinking water treatment facilities and resulted in
more than 400 000 ill people and several deaths (Bailey, Jarmey-Swan, Venter and du
Preez, 2004). This was the largest documented waterborne outbreak in United States
history. More recently, Cryptosporidium oocysts were found in water supplies in Ireland
in 2007. The water came from a lake and then underwent treatments with coagulation
and/or filtration. Heavy rainfall was suspected to have caused an increased pathogen load
in the lake due to run off from farms and Cryptosporidium oocysts were not completely
eliminated by the water treatment facilities (Pelly, Cormican, O’Donovan, Chalmers,
Hanahoe, Cloughley, McKeown and Corbett-Feeney, 2007).
Giardia has also been
associated with drinking water outbreaks and was responsible for 10.6% of drinking
water-associated outbreaks in the United States between 1997 and 2006 (MMWR, 2010).
Giardia has been involved in drinking water associated outbreaks in the US in 2007 and
in Norway in 2004 (Daly, Roy, Blaney, Manning, Hill, Xiao and Stull, 2009; Robertson,
Forberg, Hermansen, Gjerde, Alvsvag and Langeland, 2006).
2.6 FOODBORNE OUTBREAKS OF CRYPTOSPORIDIOSIS AND
GIARDIASIS
Although outbreaks of food-related cryptosporidiosis and giardiasis are less reported than
waterborne infections, oocysts and cysts are able to survive in wet or moist contaminated
food (Schlundt, Toyofuku, Jansen and Herbst, 2004). Cryptosporidium parvum was
found to be the causative agent of 1699 foodborne infection cases in England and Wales
between 1996 and 2000 (Adak, Meakins, Yip, Lopman and O’Brien, 2005). In the United
States of America Cryptosporidium was the fourth most causative agent of foodborne
infections in 2004, causing 637 infections (CDC, 2006). Food associated outbreaks of
giardiasis are rarer than cryptosporidiosis foodborne outbreaks (MMRW, 2010). Giardia
was found to be responsible for 18 foodborne outbreaks in the European Union during
2006 (EFSA, 2006) while the CDC reported 16 outbreaks in the United State between
1998 and 2007 (MMRW, 2010). Table 1 summarises reported cases of foodborne
cryptosporidiosis and giardiasis outbreaks.
Cryptosporidium oocysts have been found on the surface of raw vegetables in various
countries (Monge and Chinchilla, 1996; Robertson and Gjerde, 2001b). According to
Chaidez et al. (2005) contaminated irrigation water is one of the major sources of
contamination for fresh produce. It was found in a recent study by Macarisin, Bauchan
and Fayer (2010) that Cryptosporidium parvum oocysts were able to adhere to spinach
plants after contact with contaminated water and were also able to infiltrate the plant
through the stomatal opening. This is the first study showing attachment and
internalization of oocysts on vegetables. Fresh produce irrigated with water contaminated
with Cryptosporidium oocysts could thus become contaminated. The physical structure of
the fruit or vegetable’s surface seems to influence the amount of oocysts or cysts retained
on the product. The presence of hairy structures on the surface of the product might
increase the amount of cysts or oocysts retained on the fruit or vegetable (Armon, Gold,
Brodsky and Oron, 2002; Kniel, Lindsay, Summer, Hackney, Pierson and Dubey, 2002).
Fruits and vegetables can also become contaminated with oocysts or cysts during
production when farm workers work with soiled hands or when contaminated fertilisers
are used (Fayer, 2000). The presence of cattle near vegetable fields has also been linked
to the contamination of vegetables with Cryptosporidium (Rzezutka, Nichols, Connelly,
Kaupke, Kozyra, Cook, Birrel and Smith, 2010). Flies could be another possible source
of contamination of fruits and vegetables as viable Cryptosporidium oocysts and Giardia
cysts have been recovered from flies by Szostakowska, Kruminis-Lozowska, Racewicz,
Knight, Tamang, Myjak and Graczyk (2004). Oocysts are able to survive in the soil for
up to 12 weeks (Olson, Goh, Phillips, Guselle and McAllister, 1999) and Giardia cysts
can survive for more than 2 months in cool conditions (Bingham, Jarroll and Meyer in
1979 according to Adam, 1991). The cool and moist surface of fruits and vegetables thus
provides suitable conditions for the survival of Cryptosporidium oocysts and Giardia
cysts. The multiple possible sources of contamination indicate that sufficient viable
oocysts could contaminate fresh produce and be ingested by susceptible hosts.
Table 1: Reported foodborne outbreaks of cryptosporidiosis and giardiasis
Product
Year
Country
Organism
Infected
Reference
cases
Unpasteurised milk
2001
Australia
Cryptosporidium spp.
8
Harper, Cowell, Adams,
Langley and Wohlsen, 2002
Salad bar: whole carrots,
2005
Denmark
Cryptosporidium spp.
99
Ethelberg, Lisby, Vestergaard,
grated carrots and red
Enemark, Olsen, Stensvold,
peppers
Nielsen, Porsbo, Plesner and
Molbak, 2009
Salad bar
2008
Finland
Cryptosporidium spp.
72
Ponka, Kotilainen, RimhanenFinne, Hokkanen, Hanninen,
Kaarna, Meri and Kuusi, 2009
Parsley
2008
Sweden
Cryptosporidium spp.
21
Insulander, Jong and
Svenungsson, 2008
Sliced raw vegetables
1990
United
Giardia lamblia
26
State of
Mintz, Hudson-Wragg, Mshar,
Cartter and Hadler, 1993
America
Cold noodle salad
1985
United
Giardia lamblia
16
Petersen, Cartter, Hadler, 1988
Giardia lamblia
9
Porter, Gaffney, Heymann and
State of
America
Fruit salad
1986
United
State of
America
Parkin, 1990
Ice
1990
United
Giardia lamblia
State of
28
Quick, Paugh, Addiss,
Kobayashi and Baron, 1992
America
2.7
INDICATOR ORGANISMS
Testing environmental samples for specific pathogens can be expensive and time
consuming. Also, the detection of specific pathogens in food or water can be difficult due
to their presence in low numbers (Scott et al., 2002). This is why the practice of
monitoring indicator organisms has become widespread (Britz, 2007). An indicator
organism in food is described as a microorganism or group of them that indicate that the
food has been exposed to conditions that might introduce hazardous organisms and/or
allow their growth (DOH, 2010). Ideally, indicator organisms should have survival
characteristics similar to those of pathogens of concern and should be found in higher
number than the pathogens themselves. Indicator organisms should correlate in number to
the degree of pollution and they should not be found in the absence of pollution. Finally,
indicator organisms should be relatively safe to work with in the laboratory (Scott et al.,
2002).
Cryptosporidium oocysts and Giardia cysts are spread through contaminated faeces.
Run-off water from informal settlements, farms and effluent from faulty water treatment
plants can thus contaminate rivers and dams with Cryptosporidium oocysts and Giardia
cysts. According to Beuchat (1996), human specific enteric pathogens such as
Salmonella spp., Shigella spp., Hepatitis A virus and Noroviruses are most likely to be
found in water contaminated with human faeces. Animal faeces can also be source of
various serotypes of Salmonella, E. coli, Giardia and Cryptosporidium (Beuchat, 1996).
Also, most pathogens associated with fresh produce originate from the enteric
environment. That means that they are found in the intestinal tracts and fecal material of
humans and animals (Harris, Farber, Beuchat, Parish, Suslow, Garrett and Busta, 2003).
This is why indicator organisms are used to indicate faecal pollution and the possible
presence of pathogens.
Faecal coliforms are commonly used as indicator organisms to indicate faecal
contamination and to predict the presence of enteric pathogens (Rhodes and Kator, 1988;
Tallon, Magajna, Lofranco and Leung, 2005) since the presence of thermo tolerant
coliforms and E.coli has been found to have significant predictive values for the presence
of entero-pathogens (Horman et al., 2004). E. coli has long been used as an indicator of
faecal pollution. It meets many of the criteria of a good indicator organism as it is not
normally pathogenic to humans and is present in a higher number than the pathogens it
predicts. In warmer climates, E. coli is however able to replicate in contaminated soil
which may decrease its reliability as an indicator under such condition (Desmarais, SoloGabriele and Palmer, 2002). It has now been observed that the ecology, prevalence and
resistance to stress of faecal coliforms differ from those of many pathogens (Scott et al.,
2002). The use of other indicator organisms such as Enterococcus spp., Clostridium
perfringens and bacteriophage has thus been suggested (Scott et al., 2002; Harwood,
Levine, Scott, Chivukula, Lukasik, Farrah and Rose, 2005). The enteroccocus group is a
subgroup of faecal streptococci and has been found to be good indicators of faecal
pollution especially in marine environments and recreational waters (Griffin, Lipp,
McLaughlin and Rose, 2001). Clostridium perfringens is an enteric, gram-positive,
anaerobic, spore-forming, pathogenic bacterium and is found in human and animal faeces
(Scott et al., 2002). It has the ability to survive for a longer period of time in the
environment and has thus been used to predict the presence of viruses or remote fecal
pollution (Payment and Franco, 1993). Bacteriophages have been suggested as indicators
of enteric viruses as their morphology and survival characteristic are similar to those of
enteric viruses (Havelaar, Van Olphen and Drost, 1993).
Protozoa are more resistant to environmental stresses than bacteria. Cryptosporidium and
Giardia have a distinct resting stage, the oocyst or cyst respectively, which is able to
survive for a long time in the environment. So once water has been contaminated with
either Cryptosporidium and or Giardia, these parasites will remain viable in the water for
longer than bacteria. According to Rose and Slifko (1999), Cryptosporidium oocysts are
able to survive in river water for 176 days at 5-10˚C. Giardia cysts were shown to
remain viable for 56 days in river water (DeRegnier, Cole, Schupp and Erlandsen 1989),
while E. coli has been shown to die off in rivers after only 4 to 11 days at 4˚C (Flint,
1986).
Water contaminated with Cryptosporidium or Giardia might therefore not
necessarily test positive for E. coli due to the longer survival of these protozoa in water.
In previous studies, no correlation was found between the concentration of indicator
organisms and presence or absence of Cryptosporidium and Giardia in surface water and
water effluent from water treatment plants (Harwood et al., 2005; Horman, RimhanenFinne, Maunula, von Bonsdorff, Torvela, Heikinheimo and Hanninen, 2004). It has also
been suggested that monitoring of faecal coliforms levels on fresh produce might not
adequately reflect the occurrence of Cryptosporidium and Giardia due to coliforms’ high
susceptibility to chemical disinfection compared to the two protozoa (Harwood et al.,
2005). The monitoring of several indicators, such as Clostridium perfringens, as well as
some key pathogens was suggested as a more accurate alternative to the one-indicator
system to predict the presence of human pathogen in water (Harwood et al., 2005).
2.8
MICROBIAL SOURCE TRACKING
The role of water and food as a vehicle for transmission of cryptosporidiosis and
giardiasis is now well recognised. Cryptosporidium oocysts and Giardia cysts can
contaminate fresh produce via various routes including contaminated irrigation water
(Amahmid, Asmama and Bouhoum, 1999; Armon et al., 2002). Knowing the exact
source of contamination of fresh produce would make the management and remediation
of the pollution more effective (Simpson, Santo Domingo and Reasoner, 2002). Several
microbiological, genotypic and phenotypic techniques are available to determine the
exact source of contamination. The presence of small differences within different groups
of microorganisms are detected and then used to identify the host or environment the
organisms come from (Scott et al., 2002). Genetic methods are based on the assumption
that progeny of a specific population adapted to a specific environment will be
genetically identical and that therefore a group of organisms originating from a particular
host or environment will possess similar genetic fingerprints which will differ from
organisms adapted to different hosts or environments (Scott et al., 2002). Similarly,
phenotypic methods identify phenotypic differences between organisms focusing on a
particular trait the organisms might have acquired from exposure to different host species
or environments (Scott et al., 2002).
Foodborne outbreaks of cryptosporidiosis and giardiasis have been reported in both
developed and developing countries (Robertson and Gjerde, 2001). The exact food
responsible for outbreaks is sometime difficult to identify due to the long incubation
period and global sourcing of ingredients (Smith et al., 2006). Although 15 species and
more than 40 genotypes of Cryptosporidium have been isolated from specific hosts and
environmental samples only some species of Cryptosporidium are known to cause human
infections namely C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris, C.
suis and cervine genotype (Ruecker, Braithwaite, Topp, Edge, Lapen, Wilkes, Robertson,
Medeiros, Sensen and Neumann, 2007). Cryptosporidium hominis and C. parvum are the
most common species isolated from humans although C. hominis has been responsible
for more outbreaks than C. parvum (Smith, Caccio, Tait, McLauchlin and Thompson,
2006). Existing techniques commonly used to isolate Cryptosporidium from water and
food samples do not identify the species or genotype of Cryptosporidium present in the
sample and thus cannot accurately assess the risk for human infection (Ruecker et al.,
2007). In order to effectively assess the risk to human health associated with the presence
of Cryptosporidium oocysts in water or food, the species and/or genotypes present in the
sample must be identified (Ruecker et al., 2007). Molecular characterization of oocysts
isolated from food or water samples can also be used to determine the host faecal sources
of contamination (Ruecker et al., 2007). The most commonly found species in surface
and waste waters are C. parvum and C. hominis as well as many species of
Cryptosporidium infecting only specific animals and not pathogenic to human (Smith et
al., 2006).
2.9
SALMONELLA SEROTYPING
Salmonellas are one of the most important causes of foodborne illness worldwide and
even though most Salmonellas are regarded as potential human pathogens, their
characteristics and the severity of the illness that they cause differ between species and
serovars (Adams and Moss, 2004). Species within the Salmonella genus are differentiated
according to the Kauffman-White serotyping scheme (Garrity, 2005). The KauffmanWhite scheme describes Salmonella on the basis of their somatic (O), flagellar (H) and
capsular (Vi) antigens. The nomenclature of the Salmonella genus is different from the
nomenclature of other genera as different serovars of Salmonellas are named as if they
were distinct species (Adams and Moss, 2004). Previously, Salmonellas to be described
were given species names derived from the disease that they caused in human or animals,
e.g. Salmonella cholerae-suis. Due to the limitation of this approach, Salmonellas were
then given the name of the geographical location where they were first isolated. E.g.
Salmonella montevideo. Since 1966, this approach is only applied to serovars of the
subspecies I (S. enterica subsp. enterica) which makes up 59 % of the 2400 serovars
known and the majority (>99%) of human isolate. For the rest of Salmonella subspecies,
the serovar formula is used after the name of the subspecies. E.g. S. enterica subsp.
salamae ser. 42:g, t:-. This system now adopted internationally (Adams and Moss, 2004).
Note that the serovar is capitalised and not italicised and should be preceded by the word
“serovars” or “ser.” (Brenner, Villar, Tauxe and Swaminathan, 2000). Serovars can
further be divided into biovars and phagovars. Biovars are strains of the same serovars
that display different sugar fermentation patterns (Garrity, 2005). Biovars are determined
by the presence of absence of enzymes that enable the fermentation of specific sugars.
Phagovars are strains of the same serovars that display different sensitivity to series of
bacteriophages (Garrity, 2005).
2.10 EFFECT
OF
VEGETABLE
CRYPTOSPORIDIUM OOCYSTS
PROCESSING
ON
Foodborne infection outbreaks associated with the consumption of raw fruits and
vegetables have been increasing (Beuchat, 2002). In 2004, 190 fresh produce associated
outbreaks of food poisoning in United States were reported by the Centre for Disease
Control and Prevention (CDC) (More et al., 2007). An E. coli O157:H7 outbreak
associated with baby spinach resulted in 205 confirmed illnesses and three deaths (FDA,
2007). In 2010, Shiga toxin producing E. coli O145 was found on Romaine lettuce
infecting 30 people and causing kidney failure in 3 patients (CDC, 2010). In 2009, 235
people became infected by eating raw alfalfa sprouts contaminated with Salmonella ser.
Saintpaul (CDC 2009). In May 2011, an outbreak of Shiga toxin-producing E. coli
0104:H4 causing hemolytic uremic syndrome was reported in Europe (ECDC, 2011).
According to European Food Safety Initiative (EFSA, 2011), the outbreak has caused the
death of 49 people and infected more than 4000. The causative agent of this outbreak was
traced back to fenugreek seeds (EFSA, 2011). The protozoa cyclospora has also been
associated with several foodborne outbreaks associated with vegetables and herbs
therefore showing that protozoa can also cause fresh produce associated outbreaks
(Insulander, Svenungsson, Lebbad, Karlsson and De Jong, 2010). Lopez, Rodson,
Arrowood, Orlandi, da Silva, Bier, Hanauer, Kuster, Oltman, Baldwin, Won, Nace,
Eberhard and Herwaldt, 2001).
Processed vegetables such as frozen vegetable have also been associated, albeit more
rarely, with foodborne outbreaks (Lund, Baird-Parker and Gould, 2000). In 1997, an
outbreak of hepatitis A virus linked to the consumption of frozen strawberries infected
213 people in the state of Michigan (Hutin, Pool, Cramer, Naina, Weth, Williams,
Goldstein, Gensheimer, Bell, Shapiro, Alter and Margolis, 1999). In 2000, frozen
raspberries were implicated with a cyclosporiasis outbreak in Pennsylvania (Ho, Lopez,
Eberhart, Levenson, Finkel, da Silva, Roberts, Orlandi, Johnson and Herwaldt, 2002).
These outbreaks show that frozen fruits and vegetables can also be possible carriers of
pathogenic organisms.
Even though Cryptosporidium oocysts do not grow on food they only need to survive on
food to cause an infection. Because Cryptosporidium oocysts cannot grow or multiply on
the surface of vegetables, any Cryptosporidium oocysts present on vegetables at
consumption is there because contamination occurred during production or processing.
Irrigation water, fertilisers, contaminated soil and animal faeces can contaminate
vegetables with Cryptosporidium oocysts during production on the farm (Millar, Finn,
Xiao, Lowery, Dooley and Moore, 2002). Contaminated wash water and infected food
handlers can be sources of contamination during processing. Control of oocysts in food
can be achieved through the implementation of HACCP system specifically designed for
the elimination or reduction of viable oocysts in food (Millar et al., 2002). It is thus
important to understand the factors affecting the viability of Cryptosporidium oocysts on
fresh produce as well as on frozen vegetables and to find effective ways of inactivating
Cryptosporidium oocysts present on vegetables. The only intervention step aimed at
reducing microbial loads during the production of minimally processed vegetables is the
washing step with disinfectants. On the other hand, three main processing steps can lead
to a reduction in microbial load during the manufacture of frozen vegetables. Those are
the washing (using disinfectants), the blanching (hot water or microwaving) and the
freezing steps. No previous studies have looked at the individual as well as combined
effects of processing on Cryptosporidium viability when inoculated on vegetables. This
study will therefore investigate the effect of individual processing steps as well as the
hurdle effect of combination of steps on the viability of Cryptosporidium oocysts in
frozen vegetables.
2.10.1 EFFECT OF CHLORINE
Washing of vegetables in water baths containing disinfectants is often the only step
aimed at reducing microbial load on fresh produce during the preparation of ready-to-eat
minimally processed vegetables (Pirovani, Piagentini, Guemes and Arkwright, 2004).
Chlorine is the disinfectant mostly used at concentrations of 50 to 200 mg/L in the form
of liquid or hypochlorite salt (Sanz, Giménez, Olarte, Lomas and Portu, 2002). The
effect of chlorine on microbial load depends on the available free chlorine concentration
which is affected by various processing conditions such as initial chlorine concentration
and water-to-product ratio (Pirovani et al., 2004). The free chlorine concentration in
washing baths reduces over time as the chlorine reacts with organic compounds (Pirovani
et al., 2004). It was also observed that microbial reduction increases as the initial chlorine
concentration and water-to-produce ratio increase (Pirovani et al., 2004). The surface
structure of vegetables, such as the presence of grooves and hollows as well as the
presence of hydrophobic waxy cuticles, affects the contact between water and
microorganisms and can affect the effectiveness of the disinfectant (Adams, Hartley and
Cox, 1989). The resistant structure of Cryptosporidium oocysts makes it difficult to kill
with disinfectants (such as chlorine) commonly used in fresh produce processing. It was
found by Korich, Mead, Madore, Sinclair and Sterling (1990) that Cryptosporidium
oocysts need to be exposed to 80 ppm free chlorine for two hours to be inactivated. In
another study by Fayer (1994), Cryptosporidium oocysts were found to be infectious after
exposure to undiluted laundry bleach for 120 min. It was also found that exposure to 8 to
16 g of free chlorine per liter for 24 hours was required to kill oocysts independently of
temperature of pH (Robertson and Smith, 1992). No literature could be found on the
mechanism of action of chlorine on Cryptosporidium oocysts but Young and Setlow
(2003), suggested that hypochlorite kills Bacillus subtilis spores by damaging the spore’s
inner membrane but does not affect the DNA. Chlorine might have a similar effect on
Cryptosporidium oocysts since oocysts also have a thick cell wall like bacterial spores do.
2.10.2 EFFECT OF BLANCHING
Blanching of vegetables by immersion into hot water or use of steam is commonly used
as a pre-processing step in the manufacture of frozen vegetables (Brewer, Begum and
Bozeman, 1995). Blanching performs the following functions. It inactivates enzymes that
could cause undesirable changes during freezing storage. It enhances or fixes the green
colour of some vegetables. It induces wilting in leafy vegetables and so facilitates
packing. Finally, it reduces the number of microorganisms on the food (Splittstoesser,
Bowers, and Wilkison, 1980). This latter function of blanching will be investigated in this
study.
It has been observed that Cryptosporidium oocysts are sensitive to extreme temperatures
and studies have shown that pasteurisation treatment is sufficient to kill oocysts (Harp,
Fayer, Pesch and Jackson, 1996). When suspended in water or milk, oocysts seem to be
sensitive to heat and die when subjected to high-temperature-short-time pasteurisation
treatment of 71.7˚C for 15 s (Harp et al., 1996). Holding the oocysts at 45˚C for 20 min
was also found sufficient to render the oocysts non infective (Anderson, 1985). Blanching
of vegetables could therefore be an effective way of eliminating oocysts from vegetables
since blanching is usually carried out between 75°C and 95°C for 1 to 10 min depending
on the size of the vegetable pieces and on the type of vegetable (Barbosa-Canovas,
Altunakar, Mejia-Lorio, 2005).
The medium surrounding the microorganisms affects their sensitivity to heat and makes
them more or less resistant. The presence of water makes microbial cells less resistant to
heat and it was found that dried cells are more resistant to heat than moist cells (Jay,
2000). Denaturation of protein is the main mechanism of death by heat and occurs more
readily in the presence of water which allows for thermal breaking of peptide bonds. This
process requires more energy in the absence of water and this could explain the greater
resistance of microbial cells to heat in the absence of water (Jay, 2000). Vegetables
contain a high percentage of water e.g. broccoli contains around 91% and peppers around
92% water (Bastin and Henken, 1997). The high moisture content of fresh vegetable
might thus affect the survival of Cryptosporidium oocysts during blanching by making
the oocysts more susceptible to the heat treatment.
2.10.3 EFFECT OF FREEZING
The effects of freezing on microorganisms present on vegetables vary greatly from strain
to strain and depend on the type of freezing employed (Jay, 2000). Previous studies have
shown that the effect of freezing on Cryptosporidium oocysts is dependent on the timetemperature combination used as well as on the type of freezing employed. Fayer and
Nerad (1996) found that cryogenic freezing at -70˚C for 1 h, -20˚C for 24 h or -15˚C for
168 h was sufficient to render the oocysts non infectious. However cryogenic freezing is
not commonly used for the commercial production of frozen vegetables and the
temperature used for storage is generally above -20°C (Barbosa-Canovas et al., 2005).
According to Harrison and Croucher (1993) in Barbosa-Canovas et al., (2005), the legal
limits for cold store temperature, distribution temperature and retail display temperature
are -18ºC, -15ºC and -12ºC respectively. Slow freezing was found to be less effective
than fast freezing and 152 hours at -22˚C was required to kill 90% of oocysts in a study
by Robertson et al. (1992). The exact mechanism of action of freezing on
Cryptosporidium oocysts is unknown. Freezing affects microorganisms in various ways.
During freezing ice crystals form and deplete the amount of water available to the cells
creating a low water activity environment. The viscosity of cellular matter increases due
to the water being concentrated in ice crystal (Jay, 2000). Freezing causes losses in
cytoplasmic gases such as oxygen and carbon dioxide and causes changes in pH of
cellular matter. Freezing also causes some denaturation of protein as consequences of the
lower water content and concentration of electrolytes (Lovelock, 1957).
2.10.4 EFFECT OF MICROWAVE HEATING
Microwave blanching of vegetables can be used in the industry as an alternative to hot
water blanching during the manufacture of frozen vegetables (Kidmose and Martens,
1999; Ramesh, Wolf, Tevini and Bognar, 2002). Furthermore microwave ovens are now
found in most households and are a convenient way to cook or heat up food. Many
convenience vegetable products can be cooked at home in their packaging using
microwave ovens and then consumed directly from the package without further washing.
It is therefore important to determine the effect of microwave processing on the viability
of Cryptosporidium oocysts. The microwave radiation and generated heat can be an
effective way of killing pathogens present on vegetables if the products are microwaved
for a sufficient amount of time. It was found by Ortega and Liao (2006) that 20s in a
microwave on full power (1 100 Watts) or 30s at 650 Watts was sufficient to inactivate
Cryptosporidium oocysts if a temperature of 80ºC was reached. Due to variability in
temperature achieved by different microwave ovens, they found that the cooking time
was not a good indicator of inactivation efficiency but that the temperature reached inside
the microwave was a more accurate indicator of oocysts inactivation (Ortega and Liao,
2006). The exact mechanism of action of microwave on Cryptosporidium oocysts is
unknown but the killing effect of microwave in general is mainly attributed to the heat
generated even though non-thermal effects have also been observed (Banik,
Bandyopadhyay and Ganguly, 2003). As discussed previously, heat mainly affects
microorganisms by causing denaturation of proteins (Jay, 2000). With regards to the nonthermal effects of microwaves, it has been proposed that the microwaves cause ions to
accelerate and collide with other molecules or cause dipoles to rotate and line up rapidly
with the microwave’s rapidly alternating electric field which would cause changes in
secondary and tertiary structure of microorganism’s proteins (Banik et al., 2003).
2.11 CONCLUSIONS
Cryptosporidium and Giardia are waterborne and foodborne pathogens and can
contaminate fresh produce via various routes. Due to the poor quality of available
irrigation water in parts of South Africa, irrigation water might be a possible route of
contamination of fresh produce with Cryptosporidium and Giardia. Quality assessment
of irrigation water and fresh produce need to be carried out to determine the economic
consequences as well as potential public health risks associated with the use of irrigation
water of poor quality in South Africa. The validity of using indicator organisms to predict
the presence of Cryptosporidium and Giardia in environmental samples must also be
established.
Survival of Cryptosporidium and Giardia on fresh produce has the potential to cause
severe foodborne outbreaks. Literature available on the effect of treatments on
Cryptosporidium viability under different conditions is limited. Little information and no
previous work have been done on the combined effect of washing with chlorine,
blanching and freezing on Cryptosporidium viability.
This study investigates the
individual and hurdle effect of vegetable pre-processing and processing steps on the
viability of Cryptosporidium oocysts inoculated on green pepper.
2.12 HYPOTHESES
1. Since both Cryptosporidium and Giardia are transmitted through faeces of
infected hosts (Tzipori and Ward, 2002), the presence of faecal indicators (i.e.
faecal coliforms and E. coli) in water can be used to predict the presence of
Cryptosporidium or Giardia in water and on vegetables irrigated with such water
(Horman, 2004).
2. Because of their thick wall structure made of a double layer of protein-lipidcarbohydrate matrix (Templeton et al., 2004), Cryptosporidium oocysts have been
found to be resistant to chemical disinfectants (Lorenzo-Lorenzo et al., 1993).
Chlorine will thus not affect the membrane permeability of oocysts inoculated on
green pepper and can therefore not be expected to inactivate Cryptosporidium
oocysts present on vegetables.
Heating (Fayer, 1994; Anderson, 1985),
microwave heating (Ortega and Liao, 2006) and freezing (Fayer and Nerad, 1996)
on the other hand have been found to affect the viability of oocysts. Blanching,
microwave heating and freezing will thus alter the membrane permeability of
oocysts inoculated on peppers and render the oocysts non-infective. Treatments
combining chlorine with blanching, microwaving and/or freezing will therefore be
more effective than chlorine treatments alone.
2.13 OBJECTIVES
1. To determine simultaneously the incidence of indicator organisms, E. coli,
coliforms and Salmonella spp., as well as the incidence of Cryptosporidium and
Giardia in the Moses river in Mpumalanga, Skeerpoort river in North West and
Klip river in Gauteng during 10 consecutive months
2. To determine simultaneously the incidence of indicator organisms, E. coli,
coliforms and Salmonella spp., as well as the incidence of Cryptosporidium and
Giardia on irrigated vegetables sampled before harvest.
3.
To determine the correlation between indicator parameters (i.e. temperature, pH,
E. coli, coliforms and Salmonella spp.) and the presence of Cryptosporidium and
Giardia in irrigation water.
4. To determine the individual effects of chlorine, blanching, freezing and
microwave heating treatments on the viability of Cryptosporidium oocysts
inoculated on green peppers.
5. To determine the combined effect of chlorine and freezing treatment as well as
combination of chlorine and microwave heating treatment on the viability of
Cryptosporidium oocysts inoculated on green peppers.
CHAPTER 3: RESEARCH
______________________________________________________________________________
3.1 INCIDENCE OF CRYPTOSPORIDIUM, GIARDIA AND INDICATOR
ORGANISMS IN SOUTH AFRICAN RIVERS USED FOR IRRIGATION
OF FRESH PRODUCE
ABSTRACT
Cryptosporidium and Giardia are protozoa that can cause severe gastrointestinal infections and
can be lethal in individuals with compromised immunity. In South Africa, few studies have
investigated the incidence of Cryptosporidium oocysts and Giardia cysts in surface water used
for the irrigation of fresh produce. In this study, three rivers used for irrigation purposes from the
Mpumalanga, North West and Gauteng provinces were analysed for the presence of
Cryptosporidium oocysts, Giardia cysts, Salmonella spp., faecal indicators and physicochemical
parameters from June 2009 to April 2010. The possible relationship between faecal indicators
and the presence of Cryptosporidium oocysts and Giardia cysts was also investigated. Out of the
30 water samples analysed, 43% were positive for Cryptosporidium oocysts, 23% tested positive
for Giardia cysts and 27% were positive for Salmonella spp. No significant differences in the
prevalence of Cryptosporidium and Giardia and indicator parameters were observed between
rivers (p>0.05). Using a logistic regression model, no significant correlations were observed
between incidence of faecal indicators and the presence of Cryptosporidium oocysts and Giardia
cysts. The presence of Cryptosporidium oocysts and Giardia cysts in irrigation water indicates a
risk of infection to consumers who eat these produce as Cryptosporidium and Giardia can come
in contact and attach on the surface of fresh produce during irrigation. The use of solely faecal
indicator organism for monitoring surface water quality is not sufficient to accurately predict the
presence of Cryptosporidium and Giardia and assess the microbiological safety of irrigation
water.
3.1.1 INTRODUCTION
In South Africa, most farmers rely on irrigation water for the production of their crop as 65% of
the country does not receive enough rainfall for rain fed crop production (FAO, 2005). Surface
waters are thus widely used as a source of irrigation water for the production of fruits and
vegetables. However, studies have indicated high levels of faecal contamination and presence of
human pathogens in South African rivers (Britz, Sigge and Barnes, 2008). The use of water of
poor microbiological quality for the irrigation of fresh produce could lead to contamination of
fruits and vegetables with pathogenic organisms such as Cryptosporidium and Giardia since
contaminated water is deposited onto the surface of the crops during irrigation (Macarisin,
Bauchan and Fayer, 2010). This is of public health significance especially since the number of
fresh produce associated outbreaks is on the rise worldwide due to the increased consumption of
fresh produce, international trade and increased number of susceptible individuals (Beuchat,
2002). Contamination of fruits and vegetables with pathogenic organisms could also have a
negative impact on South African export and lead to economic losses.
Cryptosporidium and Giardia are two of the most common and resistant human waterborne
parasitic protozoa worldwide (Chaidez, Soto, Gortares and Mena, 2005). They have been
isolated from raw water, treated effluent and drinking water in South Africa (Monique
Grundlingh, Rand Water, personal communication, 2008; Kfir, Hilner, du Preez and Bateman,
1995). Cryptosporidium has been shown to attach to the surface of vegetables following
irrigation and becomes internalized making its removal difficult to achieve (Macarasin et al.,
2010). Most reported cryptosporidiosis and giardiasis outbreaks have been associated with
drinking water. However, Cryptosporidium oocysts have been found on the surface of raw
vegetables in both developing and developed countries (Robertson and Gjerde, 2001a).
Due to the socio-economic consequences the contamination of fresh produce could have, it is
crucial to determine the level of contamination of irrigation water with human pathogens. This
study determined the occurrence of Cryptosporidium and Giardia in three South African rivers
used for the irrigation of fresh produce. The cost of the isolation methods for Cryptosporidium
and Giardia is high and the recovery rate of these methods is low. It is therefore necessary to
assess the correlation between the presence of commonly used faecal indicator organisms such as
Escherichia coli and faecal coliforms and incidence of Cryptosporidium and Giardia in order to
find a reliable indicator for those two protozoa. No studies have been done in South Africa to
determine the simultaneous incidence of Cryptosporidium and Giardia and faecal indicators.
Therefore the other objective of this study was to determine whether the presence of faecal
indicators or bacterial pathogens such as Salmonella spp. can be linked to the presence of
Cryptosporidium oocysts and/or Giardia cysts in irrigation water.
3.1.2 MATERIALS AND METHODS
3.1.2.1 SAMPLING SITES
Three sites were selected: the Moses river in Mpumalanga, the Skeerpoort river in North West
and the Klip river in Gauteng (Figure 2). The sites monitored in this study were selected because
these rivers are used as irrigation water sources for the irrigation of fruits and vegetables.
Furthermore, the sites were suspected to be contaminated with Cryptosporidium and Giardia as
high levels of faecal coliforms have been found previously in these waters (Britz, Sigge and
Barnes, 2008; DACE, 2004). The presence of faecal coliforms in water indicates contamination
with faecal matter. Since Cryptosporidium oocysts and Giardia cysts are spread through
contaminated faeces (O’Donoghue, 1995), these waters could also be contaminated with the two
protozoa.
Skeerpoort River
Moses River
Klip River
Indicates river sampling site
Figure 1: Map of South Africa showing the sampling sites (Newsbank Reference Maps,
2010)
3.1.2.2 WATER SAMPLING
Water samples were taken monthly from the three sampling sites over a period of ten months
from June 2009 until April 2010. One litre water samples were collected aseptically in sterile
glass bottles for bacteriological analyses and 50 litres samples were collected into ten litres
plastic containers for Cryptosporidium and Giardia analyses. Samples were taken from the river
bank, 20 to 30 cm beneath the water surface. The samples were transported directly to the
Department of Food Science at the University of Pretoria and were analysed within 12 hours.
3.1.2.3 FRESH PRODUCE SAMPLING
Broccoli irrigated by the Loskop Dam irrigation Scheme was sampled over a period of three
weeks prior to harvest. Lettuce from Skeerpoort farm was sampled over a period of three months
at the same time as the water samples were taken. Three lettuce or broccoli heads were selected
according to accessibility from the edge of the field, transported to the lab in cooler boxes and
analysed within 12 hours.
3.1.2.4 DETERMINATION OF PHYSICOCHEMICAL PARAMETERS
The temperature of the samples was measured with a portable device (Checktemp 1, Hanna
Instrument, Inc. Woonsocket, R1, USA) when collecting the samples at the sampling site. The
pH of each water sample was measured in the laboratory using the 211 Microprocessor pH meter
(Hanna Instruments, Inc. Woonsocket, R1, USA).
3.1.2.5 ISOLATION OF CRYPTOSPORIDIUM OOCYSTS AND GIARDIA CYSTS
Cryptosporidium and Giardia were isolated using filtration, immuno-magnetic separation and
staining with fluorescein-iso-thiocyanate (FITC) and 4’,6-diamidino-2-phenylindole (DAPI)
according to the Method 1623: Cryptosporidium and Giardia in water by Filtration/IMS/FA by
United States Environmental Protection Agency (Anonymous, 2005).
Each 50 L raw water sample was filtered through a Pall Envirochek TM 1µm HV filter capsule
(Separation, Randburg, South Africa) using a pump. The Envirocheck TM filter capsule was filled
with 125 ml prepared elution buffer containing Laureth-12 (PPG Industries, Gurnee, IL), 1 M
Tris pH 7.4, EDTA and silicone antifoam agent (Merck, South Africa) and shaken on a shaker
(Grant Instrument Ltd, England) at 300 rpm.
The elution buffer was centrifuged at 1100 x g for 16 min. The supernatant was then aspirated
and discarded using pipettes. The pellet in the centrifuge tube was re-suspended by vortexing for
10 to 15 seconds. The pellet was then transferred into flat-side Leighton tube (Dehteq, Kyalami,
South Africa) using pre-rinsed pipettes.
One ml Buffer A from the IMS kit (Invitrogen Dynabeads GC Combo, Dehteq, Kyalami, South
Africa) and 1 ml Buffer B supplied with the IMS kit was added to each Leighton tube. 100 µl of
Cryptosporidium DynabeadsTM and 100 µl of Giardia DynabeadsTM were added to the each
Leighton tube.
The Leighton tubes were placed on the rotator mixer (Rotamix Atrbiotech, US) and left to rotate
at 15 rpm for 1 h at room temperature. Immuno-magnetic separation was then performed using
the MPC-1 magnet (Dehteq, Kyalami, South Africa) and MPC-S magnet (Dehteq, Kyalami,
South Africa) according to the method’s instruction (Anonymous, 2005).
Samples on microscope slides were fixed with methanol and stained with 50 µl of
Cryptosporidium FITC and 50 µl Giardia FITC stain (Davies Diagnostic Pty Ltd, Randburg,
South Africa) and incubated at 37°C for 30 min. The samples were then stained with 50 µl DAPI
working solution (Davies Diagnostic Pty Ltd, Randburg, South Africa) and left for 2 min at room
temperature.
Each slide was scanned under fluorescence microscope (Zeiss Axiovert 200, Germany) in a
systematic way at 65 x magnifications. Oocysts and cysts with apple green fluorescent cell walls
and spherical or ovoid in shape were identified as positive for FITC. The UV filter block was
then used for the DAPI examination. Oocysts and cysts that had been identified using the green
filter were then examined under UV filter for confirmation. Cryptosporidium and Giardia were
identified as DAPI positive if they exhibit up to 4 distinct sky-blue internal nuclei.
Positive control
Positive controls were used to verify the efficiency of the method. ColorSeed TM and EasySeedTM
(BTF Pty Limited, Biomerieux, Sydney, United Kingdom) were used as positive control to
determine the recovery rate of the method. Each ColorSeed TM or EasySeedTM capsule contains an
exact amount of Cryptosporidium oocysts given with the certificate of analysis. The
ColorSeedTM and EasySeedTM were used according to the manufacturer’s instruction.
Cryptosporidium oocysts recovery rate was calculated using the formula:
Cryptosporidium recovery (%) =
Cryptosporidium detected
Number of Cryptosporidium in ColorSeed
3.1.2.6
x 100
TM
as per Certificate of Analysis
NESTED POLYMERASE CHAIN REACTION (PCR)
DNA was extracted from microscope slide using Pinpoint slide DNA isolation System (Zymo
Research, USA) followed by 15 cycles of freeze-thawing (1 min in liquid nitrogen followed by 1
min at 65ºC in thermocycler) to facilitate bursting of oocysts wall and extraction of DNA.
Amplification of Cryptosporidium DNA isolated from microscope slides was performed
according to the method described by Nichols, Campbell and Smith (2003).
3.1.2.7
ISOLATION OF BACTERIAL PATHOGENS
Method ISO 4833 (International Organization for Standardisation, 1991) was used for the
enumeration of aerobic colony count on Plate Count Agar plates (Biolab, Wadeville, South
Africa) incubated at 37ºC for 48 h.
Total coliforms, faecal coliforms and E. coli were enumerated using the most probable number
method MFHPB 19 (Government of Canada, 2002). Samples were incubated in Lauryl Tryptose
(LST) broths (Oxoid LTD, Basingstoke, Hampshire, England) at 35˚C for 24 h. Positive broths
were inoculated in Brilliant Green Bile 2% broth (Oxoid, Basingstoke, Hampshire, England) and
incubated at 35˚C for 24 h. Positive BGLB tubes were inoculated into EC broth (Oxoid,
Basingstoke, Hampshire, England) and incubated in water bath at 45˚C for 24 h. E. coli was
enumerated on Eosin Methylene Blue agar (Levine) (EMB) plate (Oxoid, Basingstoke,
Hampshire, England) incubated at 35˚C for 18-24 h. E. coli colonies were confirmed on selective
E. coli/coliform chromogenic medium (Oxoid, Basingstoke, Hampshire, England) incubated at
35˚C for 18-24 h.
The presence of E. coli O157 DNA, shigatoxic E. coli and entero-hemoragic E. coli O26, O103,
O145 and H7 DNA was determined with real time PCR using the GeneSystems (Pall
GeneSystem, Bruz, France).
Salmonella spp. was isolated according to the method ISO 6579 (International Organization for
Standardisation, 1993). Enrichment was done in Rappaport-Vassiliadis (RVS) enrichment broth
(Oxoid, Basingstoke, Hampshire, England) incubated at 42˚C for 24 h and in selenite cystine
broth base (Oxoid, Basingstoke, Hampshire, England) incubated at 37˚C for 48 h. Salmonella
spp. were isolated on Phenol Red/Brilliant Green Agar and onto XLD Agar (Oxoid, Basingstoke,
Hampshire, England) incubated at 35˚C for 20-24 h. Salmonella spp. colonies were confirmed on
Salmonella chromogenic medium (Oxoid, Basingstoke, Hampshire, England). Salmonella spp.
colonies were serotyped by the ARC-Onderstepoort.
3.1.2.8
FRESH PRODUCE ANALYSIS
Cryptosporidium oocysts and Giardia cysts were isolated from fruit and vegetable samples
according to the method described by Robertson and Gjerde (2000). The fresh produce sample
was placed in stomacher bag with 200 ml of wash solution made of 50 ml of elution solution and
150 ml of water. The stomacher bag was stomached for 5 min. The wash water was then
analysed for Cryptosporidium and Giardia according to EPA method 1623 described above.
Bacterial analysis of fresh produce was done by placing 10 g of produce in 90 ml of water in a
stomacher bag. The bag was stomached and the filtrate was used for further analyses as describe
in section 3.1.2.5.
3.1.2.9
Analyses
STATISTICAL ANALYSIS
of
variance,
performed
using
Statistica
Software
for
Windows Version 7 (Tulsa, Oklohama, USA, 2003), was used to test for significant differences
in physicochemical and microbiological quality at 95% confidence interval between the different
water sites and produce. Percentages prevalence was calculated for each pathogen analysed.
Arithmetic means and standard deviations were calculated for MPN of faecal coliforms and E.
coli, temperature and pH for each sampling site.
A binary logistic regression model analysis was performed using SAS® V9.2 running under
Window XP (SP3) as supplied by SAS Institute Inc, SAS Campus drive (Cary, North Carolina).
The analysis was performed to determine whether indicator parameters, faecal coliforms, aerobic
plate counts, pH and temperature predicted the probability of the occurrence of pathogens in
water samples. The dependant variable, the pathogen (i.e. Cryptosporidium, Giardia or
Salmonella spp.), was treated as a binary variable. That means that a score of 0 was assigned
when the pathogen was not detected and a score of 1 was assigned when the pathogen was
detected. The independent variables, the indicator parameters, were continuous.
3.1.3 RESULTS
3.1.3.1 INCIDENCE OF INDICATOR ORGANISMS AND CHARACTERIZATION OF
PROTOZOA PATHOGENS IN THE KLIP, MOSES AND SKEERPOORT RIVERS
The aerobic colony counts in the Klip river ranged from 2.3 to 6 log10 cfu/ml with an average of
5 log10 cfu/ml (Figure 3). Levels of faecal coliforms and E. coli ranged from 3.2 to 5.5 log10
counts/100 ml (Figure 3). E. coli was not found in 5 of the samples despite high faecal coliform
counts. From the 10 water samples taken from the Klip river, 7 samples were positive for at least
one pathogen and 2 samples were positive for 2 pathogens (Figure 3).
Salmonella spp.,
Salmonella ser. Enteritidis and Salmonella ser. Adeoye were isolated from 2 out of the 10
samples taken from the Klip river during the summer months (Table 3). Cryptosporidium oocysts
were present in 5 samples and Giardia cysts in 2 samples taken from the Klip River.
Cryptosporidium and Giardia were isolated from the Klip river during both winter and summer
months (Figure 3). Water samples positive for Cryptosporidium oocysts were confirmed with
PCR (Figure 4)
The recovery efficiency for Giardia was significantly lower (p<0.05) than the recovery
efficiency for Cryptosporidium with 15.3% recovery for Cryptosporidium and 9.2% recovery for
Giardia. The average recovery efficiency for the 10 positive controls for the isolation of
Cryptosporidium and Giardia was lower than reported in some studies (Robertson and Gjerde,
2001b).
Aerobic colony counts in the Skeerpoort river ranged from 2.4 to 4.7 log 10 cfu/ml (Figure 3).
Faecal coliforms counts in the Skeerpoort river varied greatly between samples and ranged from
0.9 log10 /100 ml in winter to more than 9.2 log10/100 ml in summer. E. coli was isolated from 4
out of the 10 samples and was found in samples with both high and low faecal coliform counts.
The E. coli eae intimin was detected in the Skeerpoort river in March 2010 although E. coli
0157:H7 was not detected (Table 3). The eae intimin must have originated from other enterohemorrhagic E. coli (EHEC) not included in the GeneDisc analysis. None of the samples tested
positive for Salmonella spp. but 5 samples tested positive for Cryptosporidium oocysts. Giardia
cysts were found in 2 of the samples (Figure 3). Cryptosporidium oocysts were found during
both winter and summer while Giardia cysts were only isolated in summer. Cryptosporidium
oocysts were found in water samples containing low (0.9 log10 counts/100 ml) as well as high (>
9.2 log10 counts/100 ml) levels of faecal coliforms (Figure 3).
The aerobic colony counts in the Moses River ranged from 2.4 to 4.5 log 10 cfu /ml (Figure 3).
Faecal coliforms ranged from 2.5 to 4.8 log10 counts/100 ml and 7 out of the 10 samples had
faecal coliform levels above 3 log10 counts/100ml. An increase in faecal coliforms was observed
during the beginning of summer (sampling interval 4-6) which coincides with the first rainfall
after a dry winter (Figure 3). E. coli was isolated from 8 out of the 10 samples. No E.coli was
found in two of the samples for which the faecal coliforms counts were high (4 and 4.8 log 10
counts/100 ml). E. coli shigatoxin 1 was detected in the Moses river in April 2010 although E.
coli 0157:H7 was not detected (Table 3). Out of the 10 water samples analysed, 8 tested positive
for at least one pathogen and 4 samples were positive for 2 pathogens (Figure 3). Salmonella spp.
was found in 6 samples. Salmonella ser. Gaminara, Salmonella ser. Fulica, Salmonella ser.
Infantis, Salmonella subsp. I and Salmonella subsp. II were isolated from the Moses river (Table
3). Cryptosporidium oocysts and Giardia cysts were isolated from 3 of the samples (Figure 3).
l o 1g0 v a l u e s
A
1 0
9
8
7
6
5
4
3
2
1
0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
#
#
#
+
*
#
*
#
+
A C C
(lo g 1 0 c fu /m l)
F a e c a l c o lifo r m s
(lo g 1 0 M P N /1 0 0 m l)
E .c o li (lo g 1 0
M P N /1 0 0 m l)
1
2
3
4
5
6
7
8
9
1 0
T im e in t e r v a ls
B
lo g
1 0v a lu e s
#
10
9
8
7
6
5
4
3
2
1
0
+
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
A C C (lo g 1 0 c fu /m l)
#
#
#
F a e c a l c o lifo r m s
(lo g 1 0 M P N /1 0 0 m l)
+
#
E .c o li (lo g 1 0
M P N /1 0 0 m l)
1
2
3
4
5
6
7
8
9
10
T im e in t e r v a ls
C
1 0 .0
9 .0
l o g1 0 v a l u e s
8 .0
7 .0
6 .0
*
+
5 .0
4 .0
#
+
+
*
*
*
#
#
*
*
3 .0
2 .0
A C C (l o g 1 0 c fu / m l )
F a e c a l c o l i fo r m s
(l o g M P N / 1 0 0 m l )
E . c o l i (l o g
M P N /1 0 0 m l)
1 .0
0 .0
1
2
3
4
5
6
7
8
9
10
T im e in t e r v a ls
*
Positive for Salmonella spp.
# Positive for Cryptosporidium
+ Positive for Giardia
Figure 2: Aerobic colony count, faecal coliforms counts, E. coli counts and incidence of
Salmonella spp., Cryptosporidium and Giardia in the Klip (A), Skeerpoort (B) and Moses
rivers (C) for the 10 sampling intervals between June 2009 and April 2010
The two protozoa were isolated from different water samples and only one sample contained
both Cryptosporidium oocysts and Giardia cysts. Both protozoa were isolated from water that
contained both low and high levels of faecal coliforms and were found in the Moses river during
winter as well as summer months.
3.1.3.2 INCIDENCE OF INDICATOR ORGANISMS AND CHARACTERIZATION OF
PROTOZOA PATHOGENS IN BROCCOLI AND LETUCE
The aerobic plate counts for the lettuce heads were higher than those of the broccoli but not
significantly so (p=0.24) (Table 2). Aerobic plate counts from both vegetables were acceptable
according to the guideline set up by the University of Ghent for vegetables intended for raw
consumption (Uyttendaele, Jacxsens, De Loy-Hendrickx, Devlieghere, Debevere, 2010) (Table
2). Faecal coliform counts were low on both vegetables. E. coli were isolated from the lettuce but
not from the broccoli head. None of the samples were positive for Salmonella, Cryptosporidium
or Giardia.
Table 1: Indicator parameters and percentages of samples positive for pathogens for the
vegetable samples analysed.
No. of
samples
Aerobic colony
Faecal
count
coliforms /
Log cfu / g#
100 g#
E .coli / 100 g#
Broccoli
3
3.18 (1.02)
44 (74)
0 (0)
Lettuce
3
4.38 (0.98)
37 (14)
22 (22)
#
Standard deviations in brackets
M
1
2
3
4
5
Water samples
Water samples
6
7
8
9
10 M
Positive
PCRControl
Control
400 bp.
300
200
100
Figure 3: Amplified Cryptosporidium DNA on agarose gel. Lane 1-6: slides
from water samples, 7-8 positive control slides, 9 PCR positive control, 10
PCR negative control, lanes M:100 bp DNA ladder
3.1.3.3 LINK BETWEEN INDICATOR ORGANISMS AND THE PRESENCE OF
CRYPTOSPORIDIUM AND GIARDIA IN THE THREE RIVERS.
The minimum and maximum temperature of the three rivers ranged from 13.4ºC, 12ºC and
8.4ºC in winter to 22.5ºC, 27.8ºC and 26.1ºC summer for the Klip, Moses and Skeerpoort
rivers respectively. No significant differences in temperature between rivers were observed
(p>0.05) (Table 3).
The pH of the Klip river and Moses river were within normal range and varied between 6.58 and
8.02 for the Klip river and between 6.23 and 8.21 for the Moses river. The average pH of the
Skeerpoort river was significantly higher (p=0.003) than the pH of the Moses and Klip river and
ranged from 7.2 to 8.63 (Table 3).
Aerobic colony counts were on average higher in Klip river than in the Skeerpoort and Moses
river. Faecal coliform counts in Skeerpoort river were lower on average than in the Moses and
Klip river. These differences were however not statistically significant (p>0.05) (Table 3).
Fewer pathogens were isolated from the Skeerpoort river than from the other two rivers. Only
50% of the samples taken from the Skeerpoort river contained at least one pathogen and 2
samples tested positive for 2 pathogens. There were however no significant differences (p>0.05)
between the three rivers in terms of incidence of pathogens and levels of indicator organisms.
This ten month study took place over three seasons, starting in winter 2009 and finishing in
summer 2010. More samples were found positive for Cryptosporidium in summer than in winter
and spring while more positive Giardia samples were found in spring than in winter and
summer. However, those differences in incidence between seasons were not statistically
significant (p>0.05) (data not shown).
Table 2: Indicator parameters and percentage of water samples (n=30) positive for pathogens for the samples collected
between June 2009 and May 2010 from the Klip, Moses and Skeerpoort rivers
Indicator parameters
No. of
samples
Klip1
Moses2
Skeerpoort
Average
1
3
Temperature
pH4
% of samples positives for pathogen
Aerobic
Faecal
colony
coliforms /
count/ ml
100ml
E. coli /
Salmonella
100ml
spp.
Cryptosporidium
Giardia
Any
pathogen
10
18.8
7.32
122667
47590
36 990
20
50
20
70
10
20.6
7.36
5622
10740
2 640
60
30
30
80
10
20.0
8.06
8135
2670
315
0
50
20
50
30
19.76 (5.44)5
46 849
20 333
13 315
27
43
23
66
7.58
(0.59)
Salmonella ser. Enteritidis and Salmonella ser. Adeoye were isolated from the Klip river
Salmonella ser. Gaminara, Salmonella ser. Fulica, Salmonella ser. Infantis, Salmonella subsp. I and Salmonella subsp. II and E. coli
Shigatoxin 1 were isolated from the Moses river.
3
eae intimin was isolated from the Skeerpoort river
4
Anova performed between rivers (n=10). Skeerpoort pH was significantly different from Klip and Moses pH (p=0.003).
5
Standard deviations indicated in brackets
2
Table 3: Percentage of water samples from the Klip, Moses and Skeerpoort rivers (n=30)
positive for Salmonella spp., Cryptosporidium and Giardia at various levels of faecal
coliforms in samples
Faecal
coliform
count/100 ml
<100
2 (6.7)#
Salmonella
spp.
0
100-1000
7 (23.3)
1000- 10 000
>10 000
No of
samples (%)
% of positive samples
Cryptosporidium
Giardia
100
0
Any
pathogen
100
28
14
28
42
14 (46.7)
21
57
28
78
7 (23.3)
42
28
14
57
26
43
23
66
30 (100)
Total
#
Standard deviations in brackets
Table 4: Percentage of water samples from the Klip, Moses and Skeerpoort rivers (n=30)
positive for Salmonella spp., Cryptosporidium and Giardia at various levels of E. coli in
samples
E. coli
count/100 ml
No of
samples (%)
<1
12 (40)#
Salmonella
spp.
25
1-1000
6 (20)
1000- 10 000
>10 000
% of positive samples
Cryptosporidium
Giardia
25
0
Any
pathogen
50
33
50
33
83
9 (30)
22
55
44
77
3 (10)
33
66
33
66
26
43
23
66
30 (100)
Total
#
Standard deviations in brackets
Table 5: Logistic regression analysis of relationship between indicator parameters and
presence of Cryptosporidium and Giardia (p-value)
Indicator parameter
Temperature
pH
Aerobic colony count
Faecal coliforms
E. coli
Cryptosporidium
0.8193
0.4865
0.4112
0.6174
0.2141
p-value
Giardia
0.7845
0.6598
0.8302
0.6767
0.3038
Table 4 shows the percentage of positive water samples for pathogens at various levels of faecal
coliforms when looking at the three rivers together. Two water samples contained less than 100
faecal coliforms/100 ml. Salmonella spp. and Giardia were absent from those samples and only
Cryptosporidium oocysts were isolated from those 2 samples. Between 100 and 1000 faecal
coliforms/100 ml were found in 7 samples. The incidence of pathogens in those samples was
low. The highest incidence of Cryptosporidium oocysts was found in samples containing
between a 1000 and 10 000 faecal coliforms/100 ml. Giardia cysts were however found at equal
rate in samples containing between 100 and 1000 faecal coliforms/100 ml and between 1000 and
10 000 faecal coliforms/100 ml. The incidence of Salmonella spp. was the highest in samples
with faecal coliform counts above 10 000 counts/100ml. However, the overall incidence of
pathogens was lower in samples containing more than 10 000 faecal coliforms/100 ml than in
samples containing between 1000 and 10 000 faecal coliforms/100 ml although more than 50 %
of the samples containing more than 10 000 faecal coliforms/100 ml were positive for either
Salmonella spp., Cryptosporidium or Giardia.
Table 5 shows the percentage of water samples positive for pathogens at various levels of E. coli
in water samples when looking at the three rivers together. Level of E. coli of less than 1
count/100 ml was found in 40% of the samples. In those samples the incidence of pathogens was
low, and no Salmonella spp. was found. However, half of those samples tested positive for at
least one pathogen. Levels of E.coli between one and 1000 counts/100 ml water were found in
20% of the samples. The incidence of pathogen in those samples was high and 83% of the
samples tested positive for at least one pathogens. Between 1000 and 10 000 E. coli/100 ml were
found in 30% of samples. The highest incidence of Giardia cysts was observed in these samples.
The highest incidence of Cryptosporidium oocysts was observed in samples containing more
than 10 000 E. coli/100 ml.
Predictive relationships between indicators and pathogens were evaluated using binary logistic
regression. Results from each river were analysed separately by rivers or as pooled data set (all
rivers together) to determine if the concentration of any of the indicators were correlated with the
presence of Salmonella spp. Cryptosporidium or Giardia. The incidence of Salmonella spp.,
Cryptosporidium and Giardia did not correlate significantly with the any of the indicator
parameters. Aerobic colony counts, faecal coliform counts, E. coli counts, temperature and pH
had no significant effect on the presence or absence of the pathogens (Table 6).
3.1.4 DISCUSSION
Cryptosporidium oocysts, Giardia cysts and Salmonella spp. were isolated from surface water
used for irrigation of vegetables. The presence of these human pathogens in South African
irrigation water may have serious public health implications as irrigation water can be a potential
source of contamination of fresh produce as the pathogens can come in contact and attach to the
surface of the crops (Armon et al., 2002; Macarisin et al., 2010).
The aerobic plate counts and faecal coliform counts of the Klip river where higher than those of
the Skeerpoort and Moses rivers which indicates higher microbiological pollution in the Klip
river (DOH, 2010). All samples from the Klip river were above the WHO guideline for irrigation
water which limits the level of faecal coliforms to 1000 counts/100 ml (WHO, 1989) while the
Moses and Skeerpoort river had faecal coliform level above the WHO standard in many but not
all samples. This means that the Klip river has been contaminated with higher levels of faecal
matter from human or animal origin than the two other rivers although the faecal contamination
levels of the Moses and Skeerpoort were also high. The Klip, Moses and Skeerpoort rivers could
have become contaminated due to run off water from informal settlement located near the rivers
and from run off from nearby farms. The presence of high level of faecal coliforms in water
indicates faecal pollution and possible contamination of the river with human enteric pathogens
(Tallon et al., 2005). Water containing high levels of faecal coliforms should thus not be used for
irrigation purposes due to the risk of transfer of pathogen from the water to the fresh produce
(Armon et al., 2002).
Salmonella ser. Enteritidis, which was found in the Klip river, is a known human pathogen and
has been the source of foodborne human infection outbreaks in the past (Garrity, 2005;
Hennessy, Craig, Slutsker, White, Besser-Wiek, Moen, Feldman, Coleman, Edmonson,
MacDonald and Osterholm, 1996). It is the most common serotype of Salmonella reported in the
UK and most outbreaks have been associated with the use of raw eggs in food products (Adams
and Moss, 2004). Salmonella ser. Gaminara which was isolated from the Moses river is mostly
associated with poultry (Garrity, 2005). It has however been the aetiological agent of a
foodborne outbreak caused by infected orange juice (Cook, Dobbs, Hlady, Wells, Barrett, Puhr,
Lancette, Bodager, Toth, Genese, Highsmith, Pilot, Finelli and Swerdlow, 1998). The presence
of Salmonella spp. in irrigation water is a potential human health risk as pathogens can be
transferred to fresh produce during irrigation (Tyrrel, 1999). No Salmonella spp. was found in
the Skeerpoort river. This could be because the pH of the Skeerpoort river was more alkaline
than the pH of the Moses and Klip river. The optimum pH for the survival of Salmonella being 7
(Adams and Moss, 2004), Salmonella spp. might not have survived in the Skeerpoort river due to
its higher pH. The temperatures recorded did not differ significantly between rivers and were
within the growth range of enteric bacteria (Adams and Moss, 2004).
The results of this study indicate the widespread presence of Cryptosporidium oocysts and
Giardia cysts in surface water as the two protozoa were found in all three rivers. The incidence
of Cryptosporidium and Giardia was the same in the Klip and Skeerpoort rivers despite much
higher levels of faecal coliforms in the Klip river than in the Skeerpoort river. This suggests that
the use of faecal coliforms as indicator for the presence of Cryptosporidium and Giardia is not
reliable. The average incidence of Cryptosporidium oocysts in the 30 water samples was higher
than the incidence of Giardia cysts. A similar trend has been observed in Norway and Portugal
(Robertson and Gjerde, 2001b; Lobo, Xiao, Antunes and Matos, 2009). The opposite was
however observed in a study on the prevalence of Cryptosporidium and Giardia in South African
water by Kfir et al. (1995), where a higher incidence of Giardia cysts than Cryptosporidium
oocysts was recorded in surface waters. The incidences of the two protozoa obtained in the
present study are within the range of results obtained from other surveys undertaken in the UK
(Watkins, Francis, Kay and Lewtrell, 2001) and in Norway on the occurrence of
Cryptosporidium and Giardia in raw water (Robertson and Gjerde, 2001b). The presence of both
protozoa in the same sample was observed in 5 out of the 30 samples (17%) analysed. This
contrasts with the results from Kfir et al. (1995) where only 2.9% of surface water sampled in
South Africa was positive for both protozoa. However, Kfir et al. (1995) used the settling
technique for concentrating cysts and oocysts while centrifugation was used in this study. The
difference in methodology might have influenced the recovery rates.
On average, more Cryptosporidium and Giardia were isolated during spring and summer but the
two protozoa were found in the Klip and Moses rivers during both summer and winter which
suggests lack of seasonality. However, Giardia was only isolated from the Skeerpoort river
during spring and summer which coincide with the rainy season. While lack of seasonality was
reported in other studies (Robertson and Gjerde, 2001b), higher incidence of Cryptosporidium
and Giardia in surface water has been observed after rainfall in some studies as heavy rainfall
causes run off of top soil and contaminated debris into rivers (Muchiri, Ascolillo, Mugambi,
Mutwiri, Ward, Naumova, Egorov, Cohen, Else and Griffiths, 2009; Atherholt, LeChevallier,
Norton and Rosen, 1998). The sampling sites chosen for the Klip and Moses rivers happened to
be watering points for cattle in the area. This means that direct contamination of the water with
animal faeces was likely to happen throughout the year thus masking the possible effects of
rainfall and seasons on the occurrence of Cryptosporidium and Giardia. On the other hand, no
farm animals were seen in the vicinity of the Skeerpoort sampling site and Giardia was only
isolated from the Skeerpoort river in summer. The effect of rainfall on the occurrence of
Cryptosporidium and Giardia could thus be observed here. Determination of viability and
infectivity of the oocysts would be important to assess the health risk associated with the
presence of Cryptosporidium and Giardia protozoa in irrigation water.
There were no significant differences in the levels of indicator organisms and incidence of
pathogens between the three rivers. This indicates widespread contamination of surface water
with faecal matter and human pathogens independent of the location of the river.
The microbiological quality of the vegetables analysed was acceptable as the aerobic plate
counts, faecal coliform counts and E. coli counts were low and no pathogens were isolated from
the vegetables. No Cryptosporidium or Giardia was isolated from the vegetable samples despite
the high incidence of these two protozoa in the water used for their irrigation. The low bacterial
counts and absence of pathogens observed on the broccoli might be attributed to the fact that the
broccoli had been irrigated only partially with water from the Loskop Dam canal, which is
supplied by the Moses and other rivers, and the rest of the irrigation water had come from a
borehole. The absence of Cryptosporidium and Giardia from the vegetables might also be due to
the low recovery rate of the method and the possibility of false negatives cannot be ruled out.
The presence of high faecal coliform counts in the water sampled did not necessarily indicate the
presence of high levels of E. coli. It was indeed observed that E. coli was absent from some
samples which had high faecal coliform counts and was sometimes present in water samples with
low faecal coliform counts. The relationship between faecal coliform counts and presence of
pathogen or E. coli counts and presence of pathogens was thus investigated.
No predictive relationship between levels of faecal coliform or E. coli and presence of
Cryptosporidium or Giardia in surface water could be drawn from the results of this study. No
correlation was found between temperature and pH and presence of Cryptosporidium or Giardia
either. Similar poor correlation between faecal indicators and presence of pathogens has also
been demonstrated in other studies (Harwood et al., 2005). The absence of correlation between
faecal indicators and Cryptosporidium and Giardia could be due to the different rates of survival
of protozoa compared to those of bacterial faecal indicator and could also be due to the
difference in recovery rate and detection limits (Scott et al., 2002). The lower microbial density
of Cryptosporidium and Giardia in surface water coupled with low recovery rate could have lead
to failure to detect them due to sampling volume that was too small. Higher sampling volumes
have indeed been used in other studies leading to higher recovery (LeChevallier, Norton and Lee,
1991).
The lack of correlation between indicator organisms and the presence of Cryptosporidium and
Giardia suggests that the monitoring of environmental water samples with only indicator
organisms is not sufficient to accurately predict the microbiological quality and safety of the
water in terms of Cryptosporidium oocysts and Giardia cysts. Cryptosporidium and Giardia
were indeed found in irrigation water that could have been classified as of acceptable
microbiological quality if only faecal indicator counts were taken into account. The presence of
these pathogens in irrigation water in South Africa indicates the potential for human infection
acquisition through the consumption of fresh produce irrigated with those waters.
3.1.5 CONCLUSIONS
This study demonstrates the widespread presence of Cryptosporidium and Giardia in surface
water used for irrigation of fruits and vegetables in three rivers from different provinces of South
Africa. The presence of these pathogens in irrigation water has serious public health implications
as these pathogens have been shown to attach and survive on the surface of fresh produce
following irrigation with contaminated water. Commonly used feacal indicators failed to reliably
predict the presence or absence of Cryptosporidium and Giardia in the water analysed. The use
of only faecal indicator organisms for monitoring surface water quality is not sufficient to
accurately predict the presence of Cryptosporidium and Giardia and assess the microbiological
safety of irrigation water. Identification of the sources of water contamination and more
understanding of the ecology of Cryptosporidium and Giardia and their distribution in the
environment in comparison to those of indicator organisms is needed in order to identify good
predictors for the presence of Cryptosporidium and Giardia in water.
3.2 EFFECT OF CHLORINE, BLANCHING, FREEZING
AND MICROWAVE HEATING ON CRYPTOSPORIDIUM
PARVUM VIABILITY
ABSTRACT
Cryptosporidium parvum oocysts have been found on the surface of vegetables in both
developed and developing countries. Vegetables can become contaminated with C.
parvum via various routes including irrigation water. This study investigated the effect of
individual treatments of chlorine, blanching, blast freezing and microwave heating as
well as combined treatments of chlorine and freezing, and chlorine and microwave
heating on the viability of C. parvum oocysts inoculated on green peppers. The viability
of the oocysts after the treatments was assessed by determining the percentage oocysts
stained with propidium iodide (PI) using a flow cytometer. Based on the PI staining, the
chlorine treatments did not affect the viability of the oocysts. Blast freezing significantly
inactivated 20% of the oocysts. Microwave heating and blanching significantly
inactivated 93% of oocysts. Treatment of oocysts with chlorine followed by blast
freezing treatment did not affect the viability of the oocysts significantly. Treatment of
oocysts with chlorine and microwave heating were significantly more effective than
microwave heating alone and inactivated 98.1 % of the oocysts (p<0.01). The study
indicates that C. parvum oocysts are sensitive to heat (hot water blanching and
microwave heating), to some extent sensitive to blast freezing treatments but are resistant
to chlorine. The use of chlorine during vegetable processing is therefore not a critical
control point for C. parvum oocysts and should not be expected to inactivate C. parvum
oocysts present on vegetables. Consumption of raw or minimally processed vegetables
can therefore constitute a health risk as C. parvum oocysts are resistant to chlorine and
can therefore still be found viable on ready-to-eat, minimally processed vegetables.
However, cooked vegetables (in hot water or in a microwave) should be safe for
consumption regardless of the pre-treatments or processing procedures followed.
3.2.1 INTRODUCTION
The consumption of vegetables has increased worldwide. People are becoming more
concerned about their health and want to consume raw or lightly cooked vegetables
(Pollack, 2010). Also due to the busy modern lifestyle, consumers have less time to
prepare their vegetables at home and prefer to buy already washed, ready-to-eat raw
vegetables or frozen vegetables pieces that just need to be cooked briefly in hot water or
microwaved for even more convenience (Zink, 2009).
The number of foodborne infection outbreaks associated with fresh produce has however
been increasing worldwide (Rose and Slifko, 1999). Foodborne infections associated with
frozen produce have also been reported (Ho et al., 2002). Foodborne cryptosporidiosis
outbreaks have been reported in many countries and Cryptosporidium oocysts have been
found on the surface of vegetables in both developed and developing countries
(Robertson and Gjerde, 2001a; Ponka et al., 2008; Ethelberg et al., 2009). Furthermore,
during the first phase of this study, Cryptosporidium and Giardia were found in water
used for irrigation in South Africa. Since Cryptosporidium oocysts can be found in
irrigation water and on fresh produce, it is important to understand the effect each
processing step has on Cryptosporidium and whether oocysts can be killed during
washing in chlorinated water, blanching and/or freezing. Previous studies have
investigated the effect of treatments on Cryptosporidium oocysts inoculated in water or
milk (Fayer, 1994). However, little is known about the effect of vegetable processing on
Cryptosporidium or the effect of combined treatments on the survival of Cryptosporidium
oocysts associated with vegetables.
Washing of vegetables in a chlorinated water bath is often the only step aimed at
reducing microbial load on fresh produce during the preparation of ready-to-eat
vegetables (Adams et al., 1989). These products are usually sold as washed and ready to
consume without any further washing from the consumer’s side before consumption.
Furthermore, these products are often eaten raw (e.g. in salads) and not cooked. Washing
(in chlorinated water), blanching (in hot water or using microwaves) and blast freezing
steps are used in the production of frozen vegetables. Blanching is mainly used to
inactivate enzymes but this step also reduces microbial load on the produce (Brewer,
Begum and Bozeman, 1995; Archer, 2004). The initial blast freezing step is aimed at
increasing the shelf life of the product by slowing metabolic activities of the product as
well as those of the microorganisms still present on the vegetable and thus could affect
the viability of Cryptosporidium oocysts. Furthermore, at home cooking of fresh or
frozen vegetables using microwave ovens often occurs and many convenience vegetable
products are now available on retail in a microwaveable packaging. At home microwave
cooking of vegetables could therefore also have an effect the viability of
Cryptosporidium oocysts (Ortega and Liao, 2006).
This challenge test was performed on green peppers to simulate the condition of
vegetables’ surfaces. Green peppers were chosen because their smooth surface facilitated
the recovery of the oocysts after treatments and subsequent enumeration with a flow
cytometer. The selection of treatment combinations was based on the assumption that
chlorine alone would not be effective against C. parvum while the hot water blanching
treatment would be effective against C. parvum as shown in the literature. Therefore the
hurdle effect of combination of chlorine treatment with freezing or chlorine treatment
with microwave heating was investigated as little literature was available about the effect
of these treatments on C. parvum viability.
The individual effects of chlorine, hot water blanching, microwave heating and blast
freezing as well as the combined effect of chlorine and freezing, and chlorine and
microwave heating on the viability of Cryptosporidium oocysts inoculated on green
peppers were investigated during this study.
3.2.2 MATERIALS AND METHODS
3.2.2.1 EXPERIMENTAL DESIGN

The effects of 100 ppm and 200 ppm chlorine treatments on the viability of C.
parvum oocysts inoculated on green pepper pieces were compared with each other
and with the control samples.

The individual effects of 200 ppm chlorine, hot water blanching, blast freezing
and microwave heating on the viability of C. parvum inoculated on green pepper
pieces were compared with each other and with the control samples.

The effects of a 200 ppm chlorine treatment followed by a blast freezing
treatment were compared to the control samples as well as to the individual effect
of chlorine and blast freezing treatments on the viability of C. parvum oocysts
inoculated on green pepper pieces.

The effects of a 200 ppm chlorine treatment followed by a microwave heating
treatment were compared to the control samples as well as to the individual
effects of chlorine and microwave heating treatments on the viability of C.
parvum oocysts inoculated on green pepper pieces.
3.2.2.2 C. PARVUM OOCYSTS INOCULATION ONTO GREEN PEPPER
Live Cryptosporidium parvum oocysts (5x106 oocysts in 8 ml PBS) were obtained from
Waterborn Inc. (New Orleans, LA, USA). Eighty micro litres of the stock inoculums was
inoculated onto 2 g green pepper pieces previously washed with ethanol and placed
inside 5 ml sterile plastic tubes. Each inoculated piece was left to dry inside its tube at
4˚C for 1 h before being treated. Six pieces of green pepper were inoculated for each
treatment in order to have six replicates of each treatment.
3.2.2.3 EFFECT OF CHLORINE ON THE SURVIVAL OF C. PARVUM
OOCYSTS INOCULATED ON GREEN PEPPER
The period of exposure used for the chlorination treatments were chosen to mimic the
washing treatments used in the industry during the processing of frozen green pepper
strips. The effect of chlorine at two different concentrations was studied.
All glassware used was first washed in chlorine-free distilled water. Chlorine solutions,
100 ppm and 200 ppm were prepared from 3.5% hypochlorite. Chlorine, 1 ml 100 ppm,
was added to six tubes containing inoculated green pepper pieces. One ml 200 ppm
chlorine was added to another 6 tubes. After 40 s, 50 µl 10% Na 2S2O3 was added to each
tube to neutralize the free chlorine (Korich et al., 1990). Complete neutralisation of
chlorine was tested with potassium iodide starch paper (Macherey-Nagel GmbH & Co
KG, Düren, Germany).
3.2.2.4 EFFECT OF BLANCHING ON THE SURVIVAL OF C. PARVUM
OOCYSTS INOCULATED ON GREEN PEPPER
The time/temperature combination used for the blanching treatment was chosen as an
average temperature and time used to blanch vegetables in industry (Puupponen-Pimia et
al., 2003). Six plastic tubes containing inoculated green pepper pieces were placed in a
96˚C water bath for 3 min and then cooled in 4ºC water for 3 min to stop the heating
process.
3.2.2.5 EFFECT OF BLAST FREEZING ON THE SURVIVAL OF C. PARVUM
OOCYSTS INOCULATED ON GREEN PEPPER
The time/temperature combination used for the freezing treatment was chosen to mimic
the initial blast freezing treatment used in the industry during the processing of frozen
green pepper strips. Six plastic tubes containing inoculated green pepper were placed in
a blast freezer (Linde, Munich, Germany) at -20˚C for 4 min and then thawed in 4ºC
water for 3 min.
3.2.2.6 EFFECT OF MICROWAVE HEATING ON THE SURVIVAL OF C.
PARVUM OOCYSTS INOCULATED ON GREEN PEPPER
The microwave heating treatment used in this study was chosen to imitate the cooking
instructions given on the packaging of fresh vegetables and frozen vegetables in the retail
in South Africa.
Six tubes containing inoculated green pepper pieces were microwaved in a domestic
microwave oven (Daewoo model KOR_145Q, Korea) at 2450 MHz with a maximum
output power of 850 Watts for five minutes. The samples were then cooled down in 4ºC
water for 3 min to stop the heating process.
3.2.2.7 HURDLE EFFECT OF COMBINED TREATMENTS ON THE SURVIVAL
OF C. PARVUM OOCYSTS INOCULATED ON GREEN PEPPER
Chlorination and blast freezing treatment of oocysts
Six plastic tubes containing inoculated green pepper pieces were first treated with 200
ppm chlorine as described in 3.2.2.3 and then immediately frozen as described in section
3.2.2.5.
Chlorination and microwaving treatment of oocysts
Six plastic tubes containing inoculated green pepper pieces were first treated with 200
ppm chlorine as described in 3.2.2.3 and then immediately microwaved and cooled down
as described in section 3.2.2.6.
3.2.2.8 CONTROL
A control sample was used to determine the propidium iodide (PI) fluorescence emitted
by live stained C. parvum oocysts. 6 green peppers pieces were inoculated, kept at 4˚C
and then recovered and stained the same way as the treated samples.
3.2.2.9 RECOVERY OF C. PARVUM OOCYSTS AFTER TREATMENTS
Four millilitres buffer made of peptone buffer saline and 0.01% Tween 20 was added to
each tube. The tubes were mixed using a vortex for 30 s to wash the oocysts off the green
pepper pieces. The content of each tube was then transferred to a 5 ml centrifuge tube
through a 35 µm pore size nylon filter placed in a funnel. The tubes were centrifuged at
2500 x g for 10 min. The brake was set at level 025 (Cook et al., 2007). The supernatant
was aspirated with a micropipette to leave 1 ml pellet in each tube. The tubes were kept at
4˚C until staining.
3.2.2.10 STAINING
Three microlitres Cryptosporidium labeled fluorescein-iso-thiocyanate (FITC) (Davies
Diagnostic, Randburg, South Africa) was added to each tube one hour prior to analysis,
vortexed and kept in a cooler box.
Five minutes before analysis with flow cytometer, 500 ml (PI) (Beckman Coulter Miami,
Florida, USA) was added to each tube.
3.2.2.11 FLOW CYTOMETRY ANALYSIS
The viability of C. parvum oocysts after the treatments was measured by determining the
percentage cells positive for the nucleic acid dye propidium iodide with a flow cytometer.
A strong correlation has been found between oocysts infectivity and nucleic acid staining
intensity (Neumann, Gyurek, Gammie, Finch and Belosevic, 2000).
Analyses were performed on a Epic Altra flow cytometer (Beckman Coulter, Miami,
Florida) equipped with water cooled enterprise laser and using the Expo 32 MultiCOMP
software (Beckman Coulter, Miami, Florida). Cells were identified by FITC fluorescence
and forward scattering. The percentage of cells positive for both FITC and PI was
determined.
3.2.2.12 STATISTICAL ANALYSIS
Single factor Analysis of Variance (ANOVA) was used to determine whether the
individual and combined treatments had a statistically significant effect (99% confidence
interval) on the viability of Cryptosporidium parvum when compared to the control
samples. The significant level of the ANOVA test was set at p<0.01. The least significant
difference test (LSD test) was performed to determine the significant difference between
the means of the treatments. The t-test, independent by group, was performed to compare
each treatment against the control or against another treatment. Analyses were performed
using Statistica Software for Windows Version 7 (Tulsa, Oklohama, USA, 2003).
3.2.3
RESULTS
3.2.3.1 EFFECTS OF CHLORINE CONCENTRATION ON CRYPTOSPORIDIUM
PARVUM VIABILITY
The average percentage of oocysts stained by PI after being treated with 100 ppm
chlorine (10.8%) was significantly higher (p=<0.01) than the control samples (Table 8).
The percentage oocysts stained by PI after the 200 ppm chlorine treatment (7 %) was in
the other hand not significantly different from the control samples (p=0.39) (Table 8).
There was however no significant difference in the percentage of oocysts stained by PI
between the 100 ppm chlorine treatment and the 200 ppm chlorine treatment (p=0.11)
(Figure 5). Figure 6 shows the PI peak obtained with the flow cytometer after analysis of
a sample treated with 100 ppm chlorine.
Table 6: p values for t-test analysis of % inactivated oocysts after 100 ppm chlorine
and 200 ppm chlorine treatments compared to the control
p-value
100 ppm chlorine
0.032
200 ppm chlorine
0.387
% affected oocysts
Treatment
100
90
80
70
60
50
40
30
20
10
0
Control
Chlorine 100ppm
a
b
ab
Chlorine 200ppm
Treatments
Error bars on columns represent standard deviations
Average values with different superscripts letters differ significantly (p<0.01)
Figure 4: Effects of different levels of chlorine on the survival of Cryptosporidium
parvum oocysts inoculated on green pepper (n=6).
Figure 5: Percentage Cryptosporidium parvum oocysts positive for PI fluorescence
after treatment with 100 ppm chlorine.
3.2.3.2 EFFECTS OF INDIVIDUAL TREATMENTS ON THE VIABILITY OF
CRYPTOSPORIDIUM PARVUM OOCYSTS INOCULATED ON GREEN PEPPER
5.7 % oocysts included PI in the control samples. 7 % of the oocysts chlorinated with
200 ppm included PI and had thus been affected by the treatment (Figure 7). The
difference between the control samples and 200 ppm chlorine treatment samples was not
significant (p=0.39) (Table 8). Blanching and microwave heating inactivated 93.2% and
93.5% of oocysts respectively (Figure 7). Both blanching and microwave heating were
significantly more effective (p<0.01) than blast freezing to inactivate C. parvum oocysts
inoculated on green pepper when compared to the control (Table 8). A significant
percentage (p<0.01) of oocysts were affected by the blast freezing (20.1%) treatment
compared to the control group (Figure 7). Figures 8 and 9 show the PI peaks obtained
after analysis of each sample with the flow cytometer.
Table 7: p values for t-test analysis of % inactivated oocysts after chlorine,
blanching, blast freezing and microwave heating treatments compared to the
control group
Treatment
p-value
Chlorination with 200 ppm
0.387
Blanching
<0.001
Blast freezing
<0.001
Microwaving
<0.001
% affected oocysts
c
b
100
80
200ppm Chlorine
60
Blanching
40
20
Control
a
c
c
a
Blast freezing
Microw aving
0
Treatments
Error bars on columns represent standard deviations
Average values with different superscripts letters differ significantly (p<0.01)
Figure 6: Effect of chlorination, blanching, microwaving and blast freezing
treatments on the survival of Cryptosporidium parvum oocysts inoculated on green
pepper (n=6).
A
B
C
D
E
Figure 7: Percentages Cryptosporidium parvum oocysts positive for PI fluorescence
after no treatment (Control) (A), after treatment with 200ppm chlorine (B), after
blanching (C), after blast freezing (D) and after microwave treatment (E).
(1)
(2)
Figure 8: (1) analysis with flow cytometer of Cryptosporidium parvum on bivariate
dotplots of size (FS) versus green fluorescence (pmt2). The gate (a) was defined to
include all FITC fluorescent cells. (2) Percentage Cryptosporidium oocysts positive
for PI fluorescence after blanching.
3.2.3.2 HURDLE EFFECT OF COMBINED CHLORINATION AND FREEZING
TREATMENTS ON THE VIABILITY OF CRYPTOSPORIDIUM PARVUM
OOCYSTS INOCULATED ON GREEN PEPPER
The combined treatments with 200 ppm chlorine followed by blast freezing did not affect
the integrity of the C. parvum oocysts more than the individual chlorine treatment (Figure
10). Blast freezing alone was more effective than the combined treatments as there was
no significant difference in PI inclusion (p=0.77) between the control samples and the
samples that had been treated with 200 ppm chlorine and then placed in a blast freezer
(Table 9). Figure 11 shows the PI peak obtained with the flow cytometer after analysis of
a sample treated with chlorine and blast freezing.
Table 8: p values for t-test analysis of % inactivated oocysts after individual and
combined chlorine and blast freezing treatments compared to the control group
Treatment
p-value
Chlorination at 200ppm
0.387
Blast freezing
<0.001
Chlorination + blast freezing 0.766
100
% affected oocysts
90
80
70
Control
60
50
200 ppm chlorine
40
30
20
10
b
a
a
0
Blast freezing
a
200ppm chlorine + blast freezing
Treatments
Error bars on columns represent standard deviations
Average values with different superscripts letters differ significantly (p<0.01)
Figure 9: Effect of individual chlorination and blast freezing treatment or
combination of both treatments on the survival of Cryptosporidium parvum oocysts
inoculated on green pepper (n=6).
Figure 10: Percentage Cryptosporidium parvum oocysts positive for PI fluorescence
after combined treatments with 200 ppm chlorine and blast freezing.
3.2.3.3 HURDLE EFFECT OF COMBINED CHLORINATION AND
MICROWAVE HEATING ON THE VIABILITY OF CRYPTOSPORIDIUM
PARVUM OOCYSTS INOCULATED ON GREEN PEPPER
The combination of chlorination and microwave heating treatments was significantly
(p<0.01) more effective than either chlorination or microwave heating alone at
inactivating C. parvum oocysts (Table 10). Almost all oocysts (98.1%) included PI after
the combination of the two treatments (Figure 12). Figure 13 shows the PI peak obtained
with the flow cytometer after analysis of a sample treated with chlorine and microwave
heating.
Table 9: p values for t-test analysis of % inactivated oocysts after individual and
combined chlorine and microwave heating treatments compared to the control
group
Treatment
p-value
Chlorination at 200 ppm
0.387
Microwaving
<0.001
Chlorination + microwaving
<0.001
% affected oocysts
100
90
80
70
60
50
40
30
20
10
0
b
c
Control
Chlorine 200ppm
microw ave
a
a
Chlorine 200ppm + Microw ave
Treatments
Error bars on columns represent standard deviations
Average values with different superscripts letters differ significantly (p<0.01)
Figure 11: Effects of individual chlorination and microwave heating treatment or
combination of both treatments on the survival Cryptosporidium parvum oocysts
inoculated on green pepper (n=6).
Figure 12: Percentage Cryptosporidium parvum oocysts positive for PI fluorescence
after combined treatments with 200 ppm chlorine and microwave heating.
3.2.4 DISCUSSION
This study investigated the individual effects of chlorine, heat treatments (hot water and
microwave) and blast freezing on Cryptosporidium parvum oocysts inoculated on the
surface of green peppers. The combined effects of chlorine and blast freezing treatments
as well as the combined effects of chlorine and microwave heating were also
investigated. The aim of the study was to determine whether these treatments were able
to render the oocysts non infectious and whether or not they could be used to reduce the
risk associated with the presence of C. parvum on vegetables. The green peppers were
inoculated with oocysts at a concentration exceeding levels that would normally be found
on the surface of vegetables. This was done to maximize recovery and detection of
oocysts after the treatments in order to determine how effective the treatments were.
Inclusion of propidium iodide (PI) into an oocyst only occurs if the integrity and
permeability of the cell wall have been compromised. A strong correlation between
inclusion of PI and loss of infectivity as defined by loss of in vitro excystation has been
described by Campbell et al. (1992). The inclusion of PI was thus used as an indication of
loss of viability in this study. The low PI inclusion in the control samples indicates that
most C. parvum oocysts were viable prior to the treatments. Inclusion of PI in a small
percentage of the control samples can be explained by the fact that even though the
oocysts were certified viable upon delivery, some oocysts might have been damaged
during transit from the United States of America, during storage or during analysis. The
percentage of PI inclusion in the control samples was used to compare the killing effect
of the other treatments.
Less oocyst were affected by the 200 ppm chlorine treatment than by the 100 ppm
chlorine treatment. This could be attributed to the increase in pH caused by higher
concentration of chlorine (Sanz et al., 2002). Increase in pH has been found to nullify the
antimicrobial effect of chlorine (Sanz et al., 2002). It was found by Sanz, et al. (2002)
that if the pH was not controlled, chlorine’s antimicrobial action only increased with
increase in concentration up to a 50 mg/l chlorine. Further addition of chlorine up to a
concentration of 200 mg/l to the washing bath did not further decrease microbial load and
was actually less effective than 50 mg/L. The insignificant difference between the control
and the chlorine treatments showed that the addition of chlorine to washing baths during
the preparation of fresh produce would not be expected to inactivate oocysts present on
the surface of vegetables. Cryptosporidium’s resistance to chlorine has also been reported
in other studies (Korich et al., 1990; Fayer, 1994). Furthermore, as Cryptosporidium
oocysts have been shown to be internalised through the stomata and resists hours of
washing, the risk of not eliminating Cryptosporidium oocysts from contaminated
vegetables through the use of chlorinated washing bath is even greater (Macarisin et al.,
2010; Ortega, Roxas, Gilman, Miller, Cabrera, Taquiri and Sterling, 1997). These
findings have implications for the safety of minimally processed and ready-to-eat
vegetables as these products are assumed to be safe and free of pathogens by the
consumers who do not further wash the products before consumption.
Blanching of the green peppers in hot water was sufficient to kill most C. parvum oocysts
present on the inoculated green peppers. Blanching of vegetables in hot water during the
manufacture of frozen vegetables should thus be sufficient to kill Cryptosporidium
oocysts present on the surface of vegetables. At home cooking of vegetables in hot water
should also be sufficient to kill Cryptosporidium oocysts. The killing effect of heat on
Cryptosporidium oocysts has also been reported in previous studies (Harp et al., 1996;
Moriarty et al., 2005).
Blast freezing of inoculated green pepper only affected a small percentage of the oocysts.
Pre-treatment of inoculated green pepper with 200 ppm chlorine did not increase the
susceptibility of the oocysts to freezing. This could be due to the methodology used
which created different environments which affected the survival of the oocysts. Indeed,
the oocysts on green peppers undergoing solely blast freezing were dry while the oocysts
on green peppers that had been treated with chlorine were in solution. It has in fact been
observed by Kato, Jenkins, Fogarty and Bowman (2002) that Cryptosporidium oocysts in
dry soil are more resistant to freezing than oocysts in moist or wet soil. The influence of
moisture on the effect of freezing on C. parvum oocysts viability might thus have been
observed here.
These results showed that initial blast freezing of green pepper with or without prior
chlorine treatment, are not sufficient to kill C. parvum oocysts present on their surface.
Frozen vegetables that are washed or not in chlorinated water and then frozen without
any blanching steps could thus still carry live Cryptosporidium oocysts. These findings
are supported by a previous study by Fayer and Nerad (1996) who found that freezing of
oocysts in water at -20˚C for one hour was not sufficient to render Cryptosporidium
oocysts non infective and that freezing of oocysts for 24 h at -20˚C was necessary to kill
all oocysts. The effect of the initial blast freezing of vegetables on C. parvum viability
was investigated in this study and not the effect of long term frozen storage. It could
therefore be that the viability of oocysts would be affected during storage of frozen
vegetables but that is beyond the scope of this study.
Microwave heating of the inoculated green pepper inactivated most C. parvum oocysts.
These results correlate with the finding of Ortega and Liao (2006) who found that oocysts
were inactivated after exposure to microwaves at 1 100 W for 20 s or to 700 W for 45 s.
Pre-treatment of inoculated green peppers with 200 ppm chlorine for a contact time of 40
s significantly increased the efficacy of the microwave heating against C. parvum oocysts
by 4.6%. The mechanisms of action of chlorine on Cryptosporidium oocysts is poorly
documented but the spores of Bacillus subtilis have been used as a surrogate to measure
the effect of disinfectant on Cryptosporidium oocysts (Radziminski, Ballantyne, Hodson,
Creason, Andrews and Chauret, 2002). It has been observed that chlorine decreases heat
resistance in B. subtilis spores by removing proteins from the spore coat (Wyatt and
Waites, 1975). Exposure of Cryptosporidium oocysts to chlorine could thus enhance the
killing effect of microwave heating by damaging protein in the oocysts’ cell wall. It could
also be that the presence of moisture rendered the oocysts more sensitive to heat seeing as
the oocysts on green peppers undergoing solely microwaving were dry while the oocysts
on green pepper that had been treated with chlorine were still in solution. It has indeed
been observed that microorganisms are more sensitive to wet heat than to dry heat (Jay,
2000).
These results show that microwave heating can be used to kill C. parvum oocysts present
on vegetables. Microwave blanching of vegetables during the manufacture of frozen
vegetables would eliminate the risk associated with the presence of oocysts on the
produce. Similarly, at home microwave heating of vegetables should also be sufficient to
kill oocysts present on the surface of the produce.
3.2.5 CONCLUSIONS
The use of chlorine in washing water baths does not inactivate Cryptosporidium parvum
oocysts inoculated on green pepper. Therefore, chlorination alone cannot be relied on to
ensure the safety of minimally processed green peppers. Other hurdles must be used
during the manufacture of minimally processed vegetables to ensure the elimination of C.
parvum oocysts. Heating of vegetables in hot water or in a microwave is effective
against C. parvum oocysts inoculated on green peppers. Products that undergo these steps
during processing should thus not carry live C. parvum oocysts after processing. Blast
freezing only has a slight effect on the viability of C. parvum oocysts and cannot be
relied upon to kill oocysts present on fresh produce. If vegetables do not undergo any
heat treatment new hurdles must be specifically designed to target Cryptosporidium
during vegetable processing to ensure the elimination of Cryptosporidium oocysts. On the
other hand if the vegetables are blanched during manufacture or are cooked prior to
consumption, the safety risk associated with the presence of Cryptosporidium on the
vegetable is low since the oocysts are susceptible to heat and will be killed by the heat
treatment.
CHAPTER 4: GENERAL DISCUSSION
________________________________________________________________________
4.1 CRITITCAL REVIEW OF METHODOLOGY AND
EXPERIMENTAL DESIGN
The method used in this study for the isolation of Cryptosporidium and Giardia from raw
water was the method approved by the Environment Protection Agency (EPA) which is
used internationally and also locally by the Rand Water laboratories in Vereeniging. One
limitation of this method is the amount of water that can be filtered through the filter
capsule. In the official method, 10 litres are to be filtered but in this study 50 litres were
filtered to increase Cryptosporidium and Giardia recovery as no sample had tested
positive during a preliminary study in 2008 where 10 litres of water from three rivers had
been filtered and analysed (Duhain, Minnaar and Buys, 2008). While sampling larger
volume of water increased the chance of isolating the protozoa, filtering a larger volume
of turbid water caused the filter to clog up with sediment. The high amount of sediment
in some of the water samples interfered with the Immuno-Magnetic Separation (IMS) of
the oocysts and cysts and might have resulted in lower affinity of the magnetic beads
with the cysts and oocysts. Water samples with high content of sediment should have
been filtered first with a larger pore filter in order to remove larger particles and to
facilitate further analysis.
The recorded recovery rate of the method was lower than expected but different recovery
efficiencies were observed with ColorSeeds and EasySeeds. ColorSeeds and EasySeeds
are irradiated oocysts used to calculate the recovery efficiency of the detection method.
Higher recovery rate was observed when using EasySeeds compare to when using
ColorSeeds. The oocysts and cysts from the EasySeeds were more distinct and fluoresced
more strongly while ColorSeeds were difficult to distinguish from the background under
fluorescence microscope. The lower recovery could have been due to a bad batch of
ColorSeeds and the exact reason is still unknown. But this affected the recorded average
recovery rate of the method which could have been higher if EasySeeds had been used
since the beginning of the study.
Another limitation of the method 1623 is the high cost of the consumables. The cost of
the method limited the amount of samples that could be analysed during the duration of
the study. The study would have been more representative of the actual incidence of
Cryptosporidium and Giardia in irrigation water in South Africa if more samples from
more locations had been taken.
Cryptosporidium oocysts DNA was extracted from the microscope slides and an attempt
was made to replicate the extracted DNA to identify the Cryptosporidium species present
on the slides using real time Polymerase Chain Reaction (PCR) and melting curves
analysis. More than 15 species of Cryptosporidium have been identified, each associated
with a particular human or animal host, the identification of the exact species of
Cryptosporidium present in the water analysed would therefore have been of interest to
find out the source of contamination of the rivers (Fayer, 2004). C. parvum and C.
hominis are the two species most associated with human. Zoonotic infections, when
species from animal hosts are transferred to humans and are able to cause infections in
human, have however also been reported. Identification of species would have been
useful to determine whether the source of contamination of the river with
Cryptosporidium oocysts was from human source e.g. informal settlements or animal
source and if so from which animal e.g. ruminant, bird, reptile, dog, cat, etc. This
information could be used to source the problem and develop strategies to limit such
contamination in the future. This could also indicate variation in types of contamination
sources between rivers or in the contrary widespread contamination from mainly one
source, for example, mainly human faeces e.g. run-off from informal settlements. The
identification of the species isolated from the water could however not have been used to
determine whether the oocysts present in the water posed a risk of human infection or not
as all species have the potential to cause infections in human (Fayer, 2004).
Unfortunately the presence of inhibitors and low concentration of template on the
microscope slides did not allow for successful real time PCR identification. Conventional
PCR was thus used to confirm the presence of Cryptosporidium oocysts on the
microscope slides.
E. coli serotyping was performed using the GeneDisc system (Pall GeneSystem, Bruz,
France) which is an automated real time PCR system which allows for the detection of
E.coli O157:H7 DNA, shigatoxic Escherichia coli and entero-hemoragic E. coli O26,
O103, O145 and H7 DNA. A drawback of this method is that it does not allow for
detection of other E. coli apart from those listed above. E.coli spp. isolated form the
rivers were selected on selective E. coli/coliform chromogenic medium (Oxoid LTD,
Basingstoke, Hampshire, England). Selection on this chromogenic medium is based on
the production of purple colonies by E. coli producing the enzyme B-glucuronidase.
However E. coli O157:H7 does not produce this enzyme (Abbott, Hanson, Felland,
Connell, Shum and Janda, 1994). This mean that E. coli 0157:H7 colonies would appear
pink on this chromogenic media and would be mistaken for being a coliform but not E.
coli. Some samples might thus have been incorrectly identified as not containing E. coli
after confirmation on chromogenic medium and thus not serotyped any further. This
might have led to false negatives and non detection of E. coli O157:H7 in actually
positive samples and therefore underestimation of E. coli positive samples. A different
media should have been used for the isolation of all E .coli including E. coli 0157:H7.
E.coli eae virulence factor was isolated from the water but the sample did not test positive
for E. coli O157:H7 so the virulence factor must have originated from another E. coli spp.
Further serotyping could be done on the E. coli isolate to determine the exact serotypes
and pathogenicity of the E. coli present in the irrigation water.
Propidium iodide (PI) is a DNA stain that is used to determine the membrane integrity of
cells and cells with damaged membranes are considered as non viable. Considerable
correlation has been found between PI staining and loss of viability of Cryptosporidium
(Campbell et al. 1994). However more recent studies have found discrepancy between
the results obtained with PI and infectivity studies (Bukhari, Marshall, Korich, Fricker,
Smith, Rosen and Clancy, 2000). The results obtained in this study can be used as an
indication of the effect of treatments on the viability of Cryptosporidium but cannot
replace infectivity studies.
During the preliminary studies, green peppers were inoculated with 50 000
Cryptosporidium oocysts and the oocysts were then recovered from the green peppers by
stomaching in elution solution and then centrifugation as would be used when testing for
the presence of oocysts on vegetables. Not enough oocysts were recovered with this
method to allow enumeration of the oocysts with a flow cytometer because the flow
cytometer used can only detect and enumerate cells if the samples contain a high
concentration of cells of above 5000 oocysts/ ml. This showed that isolation of oocysts
from vegetables is difficult to achieve even when high numbers of oocysts are present on
the vegetables. This could explain why no field samples of broccoli or lettuce tested
positive for Cryptosporidium or Giardia while high incidence of the two protozoa had
been recorded in the irrigation water.
Green pepper was chosen as a model vegetable for the challenge tests because of its
smooth firm outer surface which does not disintegrate upon mixing in solution and
therefore facilitates the recovery of oocysts from the vegetable after the treatments. Green
peppers are sold raw as minimally processed vegetables and also frozen e.g. in stir-fry.
Green peppers undergo washing in chlorinated water and blast freezing during the
manufacture of frozen vegetables. Green pepper was thus an acceptable model vegetable
for the chlorine and freezing treatments. However, green peppers are one of the few
vegetables that are not always blanched before freezing and were therefore not the ideal
model vegetable for the blanching treatment. Frozen green peppers can however be
cooked at home in hot water on in a microwave and could therefore still be used as model
vegetable for the heat treatments. Trials had been done using broccoli as a model
vegetable since broccoli undergoes blanching during the manufacture of frozen broccoli.
But the granular structure of broccoli interfered with the recovery of oocysts after the
treatments.
Oocysts can only reproduce in vivo or in a tissue culture and cannot replicate on
commonly used microbiological media. The number of treatments that could be
investigated in this challenge study was therefore limited by the number of oocysts that
were bought from Waterborn Inc. The selection of treatments was made to simulate the
production of minimally processed vegetables, the manufacture of frozen vegetables and
the cooking of vegetables at home. The literature shows that Cryptosporidium oocysts are
resistant to chlorine but susceptible to heat treatments. It was therefore hypothesised that
the oocysts would not be affected by chlorine treatments alone but would be affected by
the hot water blanching treatments. Few studies have been done on the effect of
microwave heating on the viability of oocysts and few studies have look at the effect of
short term blast freezing on the viability of oocysts. The microwave heating and blast
freezing treatments of inoculated green peppers were not expected to fully inactivate all
oocysts inoculated on the green peppers. The effect of combined treatments with chlorine
and microwave heating and the effect of combined treatments with chlorine and blast
freezing were therefore investigated. However, since it happened that the microwave
heating had the same effect on C. parvum oocysts than the hot water blanching, the effect
of combined treatments with chlorine and hot water blanching should also have been
investigated. There were however not enough oocysts available to test the effect of these
treatments on C. parvum viability.
During the hot water blanching treatment, the tubes containing the inoculated green
pepper pieces were placed in hot water but the green pepper pieces did not come in direct
contact with the hot water. This was done to avoid losing oocysts in the water and to be
able to recover sufficient amount of oocysts for enumeration with the flow cytometer.
The temperature reached inside the tube was lower (88ºC) than the water temperature
(96º). The inactivation effect observed in this study was thus conservative and higher
inactivation rate could be expected during hot water blanching of vegetables when the
vegetables come in direct contact with the water.
4.2 CONSEQUENCES OF THE PRESENCE OF CRYPTOSPORIDIUM
IN IRRIGATION WATER AND EFFECT OF TREATMENTS ON C.
PARVUM VIABILITY
Cryptosporidium oocysts and Giardia were found on several occasions in the three rivers
tested during the duration of the field survey. This means that these protozoa are present
in water used for irrigation purposes and have the potential to contaminate vegetables in
the field and thus cause human infections.
Cryptosporidium oocysts and Giardia cysts
were found in water samples containing both high and low levels of faecal coliforms or
E. coli. Some water samples had faecal coliform counts below the WHO standard for
irrigation water and could thus be classified as acceptable in term of microbiological
quality but yet tested positive for Cryptosporidium and Giardia. No statistical correlation
was found between faecal coliform or E. coli counts and the incidence of
Cryptosporidium or Giardia. It can thus be concluded that the use of faecal coliform or E.
coli as indicator organism to predict the presence of human pathogens in river water is
not foolproof. Other organisms, like Clostridium perfringens, have been suggested to
reliably predict the presence of Cryptosporidium or Giardia in river water (Payment and
Franco, 1993). C. perfringens is a gram positive spore-former that is typically found in
soil and in the intestine of animals (Griffin et al., 2001). C. perfringens can survive for a
longer period of time than E. coli in the environment and is resistant to environmental
stresses such as heat, desiccation and ultraviolet light (Griffin et al., 2001). Its survival in
the environment is thus more similar to protozoa’s.
The concentration of Cryptosporidium oocysts in the water samples analysed was not
determined and only the presence of the parasite in water was detected. If we assume that
only one oocysts was detected in 50 litres of water, taking the 13.5% average recovery
efficiency of the method into account, this mean 7 oocysts were actually present in 50
litres of water. This number could very well be much higher considering the fact that the
recovery efficiency was lower at times and that all oocysts on microscope slides were not
counted. Many litres of water are sprayed onto the surface of vegetables during irrigation
and this is done repeatedly throughout the growing period. Cryptosporidium oocysts can
thus accumulate on vegetables and survive until harvest and possibly consumption.
Although many studies have confirmed the presence of Cryptosporidium oocysts on fresh
vegetables in both developed and developing countries, few studies have determined the
concentration of oocysts on the fresh produce (Monge and Chinchilla, 1995; Ortega et al.,
1997; Al-Binali, Bello, El-Shewy and Abdulla, 2006). In a field study in Spain, Amoros,
Alonso and Cuesta (2010), have reported an incidence of between 4 and 15
Cryptosporidium oocysts/50 g of lettuce. In Norway, Robertson and Gjerde (2001b)
analysed various vegetables for contamination with Cryptosporidium oocyst and detected
between 1 and 8 oocysts/100 g of samples. Knowing the concentration of oocysts present
on contaminated vegetables would be useful to assess the extent of the problem. However
the mere presence of viable Cryptosporidium oocysts on vegetables should be seen as a
potential health risk due to the low infectious dose of this pathogen (Okhuysen et al.,
1999).
The results of the challenge test indicate that C. parvum oocysts are resistant to chlorine
and can thus survive the washing step during the preparation of ready-to-eat minimally
processed vegetables. Cryptosporidium oocysts’ infection dose varies between species
and individual and as little as 1 oocyst is suspected to be able to cause infections in
human. Hoornstra and Hartog (2003) investigated the risk of infection associated with
ingestion of Cryptosporidium oocysts. They determined that there is a 5% probability of
developing cryptosporidiosis infection if consuming 10 oocysts.
The percentage of inactivated oocysts after the blast freezing treatment was 20%.
However, since 5.7% of the oocysts in the control samples were also stained by PI, it can
be deduced that the blast freezing treatment actually inactivated about 14% of the
oocysts. This means that to reach a final load of 10 or less oocysts on the fresh produce
with blast freezing being the only hurdle aimed at reducing the viability of
Cryptosporidium oocysts, the initial load on the produce should not be more than 12
oocysts (Figure 15).
Figure 1: Model indicating the relationship between initial level of hazard on raw
product, effect of blast freezing, and food safety risk of final product
Blanching and microwave heating treatments were more effective than chlorine and
freezing. 93% of oocysts were stained by PI after these heat treatments. By subtracting
the percentage of oocysts that were stained by PI in the control samples we can see that
the blanching and microwave treatments actually inactivated 87% of the oocysts. This
means that out of the 50 000 oocysts inoculated on the green pepper 6350 were still
viable after microwave and blanching treatments. Therefore to reach a final load of 10 or
less viable oocysts on fresh produce after hot water blanching or microwave heating the
initial load on the produce should not be more than 79 Cryptosporidium oocysts (Figure
16).
Figure 2: Model indicating the relationship between initial level of hazard on raw
product, effect of microwave heating or blanching, and food safety risk of final
product
During the manufacture of frozen vegetables, the vegetables are usually washed in
chlorinated water, blanched and then blast frozen before being stored under frozen
condition. This study did not determine the combined effect of all three treatments but
let’s assume that the vegetables are washed in chlorinated water, blanched in a
microwave oven and then subjected to blast freezing. We can use the results of the
combined chlorine and microwave heating treatments and then assume that the blast
freezing treatment has a cumulative lethal effect on the viability of the oocysts. The
combined chlorine and microwave heating treatment affected 98% of the oocysts. When
taking into account the percentage of oocysts stained by PI in the control sample we
found that 92.4% of the oocysts were actually affected by the treatments. So if 92% of the
oocysts present on the surface of the fresh produce are affected by the combined chlorine
and microwave heating treatment and a further 14% of the surviving oocysts are affected
by the blast freezing treatment, then 153 oocysts is the maximum amount of oocysts that
can be present on the vegetable before processing that can still be reduced to 10 or less
oocysts after processing (Figure 17).
Figure 3:
Model indicating the relationship between initial level of hazard on the
raw product, effect of chlorine, microwave heating and blast freezing, and food
safety risk of final product
This study was part of a bigger national research project investigating the microbiological
quality of irrigation water all over South Africa. The combined results of the various
studies are alarming as high levels of faecal contamination and human pathogens have
been found in most water tested (Britz et al., 2008). The presence of Cryptosporidium in
rivers used for irrigation purposes is of even higher concern due to oocysts’ resistance to
chlorine. Cryptosporidium oocysts can be transferred from irrigation water to fresh
produce and then survive the washing step in chlorinated water. This would lead to the
presence of viable Cryptosporidium oocysts on the finished product. This is of particular
concerns for vegetables eaten raw. Ready-to-eat minimally processed vegetable
manufacturers must be aware of the risk and implement strategies targeting specifically
Cryptosporidium oocysts to ensure the safety of the finished product. The presence of
Cryptosporidium oocysts on vegetables that are cooked or where blanching is used as a
pre-treatment is not of as much concern as Cryptosporidium oocysts are susceptible to
heat and boiling of vegetables or microwave heating on full power will kill the oocysts.
As little information is available on the occurrence of Cryptosporidium oocysts and
Giardia cysts on vegetables in South Africa, more testing of vegetables from the field to
the retailers’ shelf is require to determine the extent of the contamination and evaluate the
potential health risk to the consumers.
Due to the high cost and complexity of the method for the isolation of Cryptosporidium
and Giardia, water samples in South Africa are seldom tested for their presence.
Indicator organisms are used to indicate contamination with faecal matter and possible
presence of human pathogens. However as this and other studies have shown, little
correlation has been found between levels of faecal coliforms or E. coli and the presence
of Cryptosporidium and Giardia. Therefore, more research needs to be done to find
suitable indicator organisms to predict the presence of Cryptosporidium and Giardia in
surface water and on fresh produce.
CHAPTER 5: CONCLUSIONS AND
RECOMMENDATIONS
High levels of feacal coliforms and E. coli were observed in the three rivers monitored
during this study. The levels of faecal coliforms in most of the samples were above the
WHO standard for irrigation water. These alarming results indicate heavy contamination
of the rivers with faecal matter.
Cryptosporidium oocysts were isolated from 43% of the water samples and Giardia cysts
were found in 23% of the samples. These results show a widespread presence of
Cryptosporidium and Giardia in surface water used for irrigation purposes. This is of
public health concern as these protozoa can be transferred to vegetables during irrigation
and survive on the fresh produce (Macarisin et al., 2010).
No significant correlation was established between levels of faecal coliforms or E. coli in
the water and presence of Cryptosporidium oocysts and Giardia cysts. Faecal coliforms
and E. coli are thus not good predictors of the presence of Cryptosporidium and Giardia
in water. The first hypothesis must therefore be rejected and faecal coliforms or E. coli
should not be used as the sole indicators of water quality. The link between the presence
of Cryptosporidium and Giardia and other indicator organisms, such as Clostridium
perfringens, must be investigated to establish a suitable indicator for the presence of
Cryptosporidium and Giardia in water.
The second hypothesis can be accepted. Cryptosporidium oocysts are resistant to chlorine
at levels used in the industry for the washing and disinfection of ready-to-eat minimally
processed vegetables. This is a serious concern since oocysts can thus survive on
vegetables during processing and still be viable on the finished product. Blast freezing
affects the viability of oocysts only slightly and cannot be relied upon to render a
contaminated product safe. More research is required to determine the survival of
Cryptosporidium during frozen storage. Cryptosporidium oocysts are killed by blanching.
There is therefore little risk of infection from consuming frozen vegetables that have been
blanched during manufactured or that are cooked in boiling water prior to consumption.
Microwave heating is also effective against Cryptosporidium oocyst. Cooking of fresh or
frozen vegetables in a microwave should thus be sufficient to inactivate oocysts present
on the vegetable.
In conclusion, the results of this study indicate the presence of Cryptosporidium oocysts
and Giardia cysts in irrigation water in South Africa and confirmed the resistance of
Cryptosporidium oocysts to chlorine. The potential therefore exists for acquisition of
Cryptosporidium oocysts and/or Giardia cysts through the consumption of contaminated
fresh or minimally processed vegetables. These findings indicate possible risk for the
food safety of South African fresh and minimally processed produce and indicate the
need for further research in this area.
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