ABUNDANCE OF VIRUSES IN WATERS IN
SOUTHWEST KAMPALA, UGANDA
By
Joyce Mujunga Nakyazze

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Environmental Engineering – Master of Science
2017

ABSTRACT
ABUNDANCE OF VIRUSES IN WATERS IN SOUTHWEST KAMPALA, UGANDA
By
Joyce Mujunga Nakyazze
The prevalence of waterborne viral diseases is still a major problem in developing
countries like Uganda. However, little is known of the viral pathogens that cause diseases. This
research aimed to study the abundance of waterborne adenovirus, rotavirus, enterovirus and
hepatitis A virus in the southwest region of Kampala, Uganda. Fifteen samples were collected
during the summer of 2016 from five sampling locations that include: Wastewater Treatment
Plant (WWTP) both influent and effluent, Nakivubo channel (upstream and downstream of the
WWTP) and Nakivubo swamp. The sequence of analytical methods used include: collection and
concentration of viruses using Argonide cartridge filters, elution of viruses with buffered beef
extract, RNA and DNA extraction, quantification of prevalent viruses using quantitative
Polymerase Chain Reaction method (qPCR). The concentrations of the viruses ranged from
5.79×101 to 3.91×107 copies/L. Adenovirus concentrations were relatively high, 5.45×105 to
1.17×107 copies/L at all the sampling locations. High concentrations of adenovirus in these
waters may be due to the high persistence of these viruses in the environment. Enteroviruses
predominated with a maximum concentration of 3.91×107copies/L over other viruses of interest.
Human exposure to such pathogens through consumption of contaminated water and food can be
harmful. These data are paramount for estimating the risk from exposure to microorganisms.

ACKNOWLEDGEMENTS
My sincere gratitude goes to my advisor Dr. Irene Xagoraraki and Dr. John B. Kaneene
for their mentorship and research ideas during my master’s program. I would like to thank Dr.
Mantha Phanikumar for serving on my graduate committee. I am also grateful for Dr. Noah
Kiwanuka for his unwavering support during this research.
I am appreciative of my lab mates, Evan O’Brien and Huiyun Wu who received and
processed the samples during the summer of 2016.
I acknowledge the financial support of this research made by:
1) The MasterCard Foundation Scholars Program-MSU
2) Institute of International Health-MSU
3) College of Engineering-MSU
4) College of Veterinary Medicine -MSU

iii

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................................... vi
LIST OF FIGURES ...................................................................................................................... vii
CHAPTER 1 - MICROBIAL WATER POLLUTION IN KAMPALA, UGANDA
(LITERATURE REVIEW)............................................................................................................. 1
1.1

Introduction ...................................................................................................................... 1

1.2

Water supply and Sanitation Practices in Kampala ......................................................... 2

1.2.1

Millennium Development Goals ............................................................................... 2

1.2.2

Drinking Water Sources ............................................................................................ 3

1.2.3

Human Excreta Disposal........................................................................................... 3

1.2.4

Wastewater Management .......................................................................................... 3

1.3

Water Quality Studies in Kampala ................................................................................... 4

1.4

Waterborne Disease Burden ........................................................................................... 10

1.4.1

Rotavirus Diarrhea .................................................................................................. 10

1.4.2

Cholera .................................................................................................................... 10

1.4.3

Dysentery ................................................................................................................ 11

1.4.4

Cryptosporidiosis .................................................................................................... 11

REFERENCES ............................................................................................................................. 13
CHAPTER 2 - ABUNDANCE OF VIRUSES IN WATERS IN SOUTHWEST KAMPALA,
UGANDA ..................................................................................................................................... 16
2.1

Introduction .................................................................................................................... 16

2.2

Materials and Methods ................................................................................................... 18

2.2.1

Sample Collection ................................................................................................... 18

2.2.2

Sample Processing .................................................................................................. 19

2.2.2.1

Elution of Argonide Cartridge Filters .............................................................. 19

2.2.2.2

Flocculation ..................................................................................................... 20

2.2.2.3

Microbial Removal .......................................................................................... 20

2.2.3

DNA Extraction ...................................................................................................... 21

2.2.4

Virus Quantification................................................................................................ 21

2.3

Results ............................................................................................................................ 24

2.4

Discussion ...................................................................................................................... 29
iv

2.5

Conclusion and Recommendations ................................................................................ 33

APPENDIX ................................................................................................................................... 34
REFERENCES ............................................................................................................................. 40

v

LIST OF TABLES

Table 1-1: Water quality studies in Kampala................................................................................. 9
Table 1-2: Waterborne disease burden in Kampala ..................................................................... 12
Table 2-1: QPCR cycling conditions used for detection of targeted viruses ............................... 22
Table 2-2: Primers and probes used for detection of targeted viruses ......................................... 23
Table 2-3: Mean genomic copy concentrations of viruses .......................................................... 24
Table 2-4: Number of samples that tested positive per qPCR runs ............................................. 25
Table A-1: Sample data sheet for fieldwork…………………………………………………… 35
Table A-2: Field data sheet for sampling carried out on 07/12/2016……………………….......36
Table A-3: Field data sheet for sampling carried out on 07/25/2016……………………….......37
Table A-4: Field data sheet for sampling carried out on 08/08/2016……………………….......38
Table A-5: Field photos for research work carried out between 07/12/2016- 08/08/2016…......39

vi

LIST OF FIGURES

Figure 1-1: Map of Kampala divisions .......................................................................................... 2
Figure 2-1: Sampling locations in Kampala ................................................................................ 18
Figure 2-2: Water flow direction across sampling locations ....................................................... 19
Figure 2-3: Distribution of viruses at sampling locations: a) Rotavirus b) Hepatitis A .............. 26
Figure 2-4: Distribution of viruses at sampling locations: c) Enterovirus d) Adenovirus ........... 27

vii

CHAPTER 1 - MICROBIAL WATER POLLUTION IN KAMPALA, UGANDA
(LITERATURE REVIEW)

1.1 Introduction
Kampala, the capital city of Uganda is located 8km north of Lake Victoria and
approximately 42 km north of the Equator. The capital city originally situated on seven hills,
has now expanded onto 24 hills surrounded by wetland valleys. Kampala has five administrative
and political divisions: Central, Makindye, Rubaga, Kawempe and Nakawa (Figure 1-1) and
serves as a political, administrative, commercial, and educational center. Due to the functions
that the city serves, the population has increased from approximately 1,189,150 in 2002 to
1,516,210 in 2014 (UBOS, 2014). The rapid increase in population is mainly attributed to ruralurban migration in search of better living standards and this has resulted into settlements
(slums) which accommodate more than 50% of the city’s population (UN Habitat, 2007). The
settlements face challenges of poor sanitation and hygiene that have exacerbated over the years
as more people move into the city (UN Habitat 2007, Kulabako et al. 2007, Isunju et al. 2011 &
Tumwebaze et al.2013). This has resulted into contamination of water sources and consequent
outbreaks of diseases such as cholera and typhoid including diarrheal cases among children
below the age of 5 years in Kampala (Odiit et al. 2014).

1

Figure 1-1: Map of Kampala divisions
1.2 Water supply and Sanitation Practices in Kampala
1.2.1 Millennium Development Goals
The World Health Organization (WHO) reports that from 1990 to 2012, 2.3 billion
people worldwide had access to improved drinking water sources while 2 billion people gained
access to improved sanitation facilities (WHO&UNICEF, 2015). The statistics showed progress
on meeting the Millennium Development Goals (MDGs) on drinking water and sanitation
facilities targets, however, access to improved sanitation facilities is still lacking especially in
countries of Sub-Saharan Africa, South East Asia and South and Central America.
Uganda, like any other developing country still faces challenges in meeting MDGs on
improved sanitation. By 2015, Uganda had not met the MDG target on sanitation with only 29%
of the urban population having access to improved sanitation facilities such as flush/pour toilets
and ventilated improved pit latrines (VIP) (WHO& UNICEF 2015). However, concerning the
MDG on drinking water, Uganda by 2015 had met its target whereby 96% of the urban
population had access to improved drinking water sources like protected springs and wells,
2

piped water.
1.2.2

Drinking Water Sources
In Kampala, approximately 60% of the population access water from standpipes and 30%

through protected springs and wells (Kulabako et al., 2010). A small percentage of 10% has
private connections due to the high costs of installation and maintenance of such connections.
Protected springs and wells are some of the main water sources for urban centers like Kampala,
however, studies have shown that more than 80% of the protected springs and wells are
contaminated with fecal matter (Byamukama et al., 2000, Haruna et al., 2005, Fuhrimann et al.,
2015). This poses a health risk to city dwellers.
1.2.3

Human Excreta Disposal
Traditional pit latrines are the major disposal method of human excreta in Kampala

(75%) , 10% use ventilated improved pit latrines (VIP) 10% use flush toilets and 5% practice
open defecation (NWSC 2004 ,Okot-Okumu & Oosterveer, 2010). The lack of space to construct
the latrines and low-income levels forces people to share pit latrines (Kwiringira, Atekyereza,
Niwagaba, & Gunther, 2014). This presents a challenge and puts the population at risk of
experiencing diseases due to poor hygiene and sanitation since the owners undertake the
maintenance of the shared toilets.
1.2.4

Wastewater Management
With approximately 10% of the urban population connected to the sewer system (NWSC,

2004), most of the residents in Kampala dispose of the wastewater in open channels and space.
This is mainly caused by a lack of financial resources and little space for construction of sewer
systems (Kulabako et al., 2010) especially among 60% of the city’s population that resides in

3

slum areas of the city (UN Habitat,2007). A case study by Kulabako et al., 2010 conducted in
Bwaise III, an impoverished area in Kampala revealed that most of the residents dispose the
greywater in open drains (37%) whereas 23% use both the open drains and open space. The rest
of the population in the area also disposes the greywater in open space (23%), soak pits (3%).
Poor disposal of wastewater affects the aesthetics of the surrounding. The wastewater
consequently finds its way into surface water bodies and hence comprising the water quality of
such water sources.
1.3 Water Quality Studies in Kampala
Studies conducted in Kampala have revealed high contamination of water sources like
Lake Victoria, wells and protected springs (Table 1-1), with bacteriological characteristics being
the major focus while the viral characteristics of the water less concentrated on. The
concentrations of bacteria in water exceed both Uganda National Bureau of Standards (UNBS)
levels and WHO standards (0 CFU /100 ml).
Lake Victoria, the main water supply to Kampala and surrounding areas has been
subjected to high levels of pollution arising from poor sanitation practices at its shores. A study
conducted by Muyodi et al., 2009 revealed high levels of water contamination at 10 sampling
sites that include lakeshores, boreholes and shallow wells around Lake Victoria. The
concentration of total coliforms ranged from 110-20.05×103 CFU/100mL and for fecal
coliforms ranged from 3-2.88×103 CFU/100mL. The water quality parameters were correlated
with prevalence of waterborne diseases such as cholera, dysentery and typhoid. The occurrence
of the water borne diseases was higher in the wet season than in the dry season due to the
deposition of fecal matter into the shallow wells and lakeshores.

4

A study conducted by Howard et al., 2003 to determine the variation of water quality in
shallow protected springs in Kampala revealed a strong correlation between short-term rainfall
events and degree of contamination of the springs. 25 shallow protected springs in both high and
low density settlements were monitored for thermotolerant coliforms and fecal streptococci over
a period of 12 months, April 1998 to April 1999. Thermotolerant coliforms and fecal
streptococci were enumerated using membrane filtration, with growth on lauryl sulphate broth
and enterococcus agar at 44oC respectively. Results showed that most of the protected springs
were contaminated with fecal matter, since the values for the indicator coliforms varied between
< 1 to 23×103 CFU/100ml, values that are above the WHO standards of 0 CFU/100ml. The
median number of coliforms displayed a strong relationship with rainfall events of 24 hour and
48 hour periods compared to the monthly rainfall events. This could be attributed to the “first
flush effect “, where rapid changes in water quality occurred after early rains. The contamination
of the protected springs poses a health risk to the 60% of the population in the city that depend
on springs for domestic water.
Kulabako et al., 2007 studied the quality of the shallow groundwater in Bwaise III, a
slum area in Kampala. Samples from 9 monitoring wells and the protected spring were analyzed
for thermotolerant coliforms and fecal streptococci for 19 months during the wet and dry seasons
of Kampala in 2003. The membrane filtration technique with lauryl sulphate broth and
enterococcus agar analyzed thermotolerant and fecal streptococci respectively. High levels of
fecal contamination were observed in the groundwater with median values of 126×103 CFU
/100ml and 154×103 CFU/100ml obtained for thermotolerant coliforms and fecal streptococci
respectively. The microbial contamination of the groundwater was due to poor waste disposal in
the area, poorly maintained and protected springs and hydrogeological characteristics of Bwaise

5

III such as high permeability of the soil and high water table.
Contamination of protected springs remains a challenge in both low and high-density
settlements of Kampala City as studied by Nsubuga et al., 2004. Eight protected springs in
Makindye and Kawempe divisions were observed for fecal coliforms and fecal streptococci,
once for five successive weeks in the dry and wet seasons. Fecal coliforms and fecal streptococci
were isolated and counted using membrane filtration, with lauryl sulphate broth and
enterococcus agar as test media at 44oC respectively. Protected springs in both the low and high
density areas had high numbers of fecal coliforms and fecal streptococci bacteria, with values
ranging from 3-18.3×103 CFU/100ml and 2-2.76×103 CFU/100ml respectively. The protected
springs in high-density settlements were more polluted than those in the low-density settlements
both in the dry and wet season. The highest number of coliforms recorded was 18.3×103
CFU/100ml in high-density settlements and 382 CFU/100ml for the low-density settlements. The
levels of coliforms in the water were above both the Uganda National Bureau of Standards
(UNBS) levels and WHO standard of 0 coliforms in every 100ml water sample. The presence of
coliforms in water serves as indicator of fecal contamination.
Slum areas like Kisenyi and Katwe face similar water quality issues like areas such as
Bwaise, in Kampala Uganda. Haruna et al., 2005 observed high levels of water contamination in
10 springs of Kisenyi and Katwe areas. Sampling was carried out biweekly from December
2001-March 2002 and the water samples were analyzed for total coliforms, fecal coliforms and
fecal streptococci using the membrane filtration method. The median values were as high as
7.9×103 CFU/100ml for total coliforms, 4.050×103 CFU/100ml for fecal coliforms and
1.130×103 CFU/100 ml for fecal streptococci. Water contamination poses health risks to
populations in such areas that depend on springs for domestic water.
6

Fuhrimann et al., 2015 studied the microbial contamination of water in the Nakivubo
wetland area in Kampala. Twenty-three sites at Nakivubo channel, Nakivubo wetland,
community areas and at the shore of Lake Victoria were sampled for thermotolerant coliforms
(TTC), Escherichia coli (E.coli) and Salmonella spp. Water samples were taken from the sites
for 8 weeks during the rainy season of October- December 2013. TTC and E.coli were detected
using the membrane filtration, with membranes incubated on lauryl sulphate broth at 44 oC and
37oC respectively. Salmonella spp were analyzed using the membrane filtration method, with
membranes incubated on xylose lysine deoxycholate agar at 37oC . The analyses showed that
TTC, E.coli and Salmonella spp were present in the water at all the sampling locations. The
mean concentrations of TTC, E.coli and Salmonella spp ranged from 3.7 to 4.3×106, 1.3 to
9.9×104 and 1.3 to 3.8×102 CFU/100ml respectively. High concentrations of the indicator
bacteria were generally detected in Nakivubo channel and wetland and the lowest concentrations
were detected at the shores of Lake Victoria. The water at all sampling sites was contaminated
with fecal matter as indicated by the concentrations of the bacteria in the water.
A study conducted by Katukiza et al., 2013 in the slum areas of Bwaise III concluded that
viral water contamination poses too a health risk to the surrounding communities. The viruses
were concentrated using the glass wool method and detected using Real time quantitative
polymerase chain reaction (qPCR) for DNA viruses. Rotavirus concentration ranged from
7.36×10-1 to 1.87×102 copies/mL with the maximum concentration obtained from surface water.
Human Adenovirus F and G were found to be prevalent in the water with the maximum
concentration of 7.62×10-3 copies/mL. The water samples tested negative for Hepatitis E virus
while few samples (15.4-27.2%) tested positive for Hepatitis A (7.4×10-1-6.87×10-1 copies/mL).
The surface water was more polluted than the grey and ground water. This could be attributed to
7

the poor solid waste, human excreta and wastewater management practices in slums such as
Bwaise III. Diarrheal diseases caused by rotavirus are the major killers of children below the age
of five.
Similarly, Chung et al., 2013 observed relatively high concentrations of rotavirus in grey
water and surface water in Bwaise III. The rotavirus concentrations varied between 2.22 to 2.91
TCID50/ml.

8

Table 1-1: Water quality studies in Kampala
Study no.

Water Source

1

Lake Victoria

2

Protected Springs

3

Protected Springs

4

Wells

5

Overall Results
TTC 110- 20.05× 103 CFU/100ml
FC 3-2.88× 103 CFU/100ml

Reference
Muyodi et al.,2009

TTC & FS <1-23× 103 CFU/100ml

Howard et al., 2003

TTC <0-10× 103 CFU/ 100ml
FS <0-8.3× 103 CFU/100ml
TTC <0-162× 106 CFU/ 100ml
FS <0-176×106 CFU/100ml

Kulabako et al., 2007

Protected Springs

FC 3-18.3× 103 CFU/100ml
FS 2-2.76× 103 CFU/100ml

Nsubuga et al., 2004

6

Protected Springs

FC 1-60.8× 103 CFU/100ml
FS 0- 3×103 CFU/100ml

7

Wetland (wastewater)

TTC 4.0× 102 - 2.2× 108CFU/100ml,
EC 1.0× 102 -7.9× 103 CFU/100ml

Fuhrimann et al.,2014

8

Drainage Channel
( wastewater)

TTC 1.2× 103-1.8× 108 CFU/100ml,
EC 8.4× 102 -9.0× 107CFU/100ml

Fuhrimann et al., 2014

Unprotected spring

7.62× 10-3 copies/mL
HAdV 1.35× 10-1 copies/mL , RV
1.45-3.32× 10-1 copies/mL , HAV
7.4×10-1 - 6.87× 10-1 copies/mL

Open grey water
tertiary drains
9

Katukiza et al.2014
Open storm water
drainage channel

10

Haruna et al., 2005

Grey water & surface
water

HAdV 1.53-5.32× 10-2 copies /mL ,
RV 7.36× 10-1 -1.87× 102 copies
/mL
RV 2.22- 2.91 TCID50/ml.

Chung et al.2013

TTC- Thermotolerant Coliforms, FC-Fecal Coliforms, FS- Fecal Streptococci, EC- Escherichia coli,
HAdV- Human Adenovirus, RV- Rotavirus, HAV- Hepatitis A

It is evident that water supplies in Kampala are highly contaminated with fecal matter.
Consumption of contaminated water poses a health risk to the city population. Therefore,
preventive measures like improved sanitation practices and adequate treatment of drinking water
are paramount in preventing the outbreak of waterborne diseases.

9

1.4 Waterborne Disease Burden
Kampala city has experienced diseases such cholera, typhoid, dysentery, rotavirus
diarrhea and cryptosporidiosis over the years (Table 1-2). The outbreak or occurrence of such
diseases is mainly attributed to unsafe water supplies, poor hygiene and sanitation practices.
1.4.1

Rotavirus Diarrhea
Children under the age of 5 mainly suffer from diarrhea which accounts for 1 in 9 child

deaths (CDC). Rotavirus is the leading cause of acute diarrhea and causes about 40% of
hospitalizations in children under five. Most diarrheal associated diseases are attributed to unsafe
drinking water, insufficient hygiene and inadequate sanitation (Bwogi et al., 2016). In Kampala,
Rotavirus diarrheal cases have been reported at hospitals such as Mulago Hospital in periods
between July 2006-June 2008 where 39% of the stool samples of children under the age of 5
tested positive for rotavirus (Mwenda et al., 2010). Odiit et al., 2014 conducted a study at
Mulago hospital between July 2008-December 2012 which also revealed a high prevalence of
acute rotavirus diarrhea among children <5 years. Also, 37% of children under the age of five at
four hospitals in central Uganda had severe diarrhea (Bwogi et al., 2016). The three studies
attributed the diarrhea outbreaks to the presence of rotavirus in stool specimens.
1.4.2

Cholera
Cholera, another common waterborne disease caused by bacteria Vibrio cholerae affected

residents of Namuwongo, an informal settlement located in Makindye division in 1995. Six
deaths were reported during the outbreak though other cases went unreported (Sengooba et.al
1995). The outbreak was attributed to poor sanitation practices like open defecation and
consumption of unboiled spring water. Other slum areas like Bwaise III in Kawempe Division
have also been affected by cholera. 23 cases were reported at Mulago hospital between October
10

2007-March 2008 (Kulabako et al., 2010).
1.4.3

Dysentery
Dysentery is caused by a number of parasites such as viruses, bacteria and protozoa.

Symptoms such as bloody diarrheal stools, abdominal pains manifest in the patients and if left
untreated may lead to death. Dysentery also remains a major threat in slums of Bwaise III, 42
and 109 cases were reported at Kawempe Health Centre and Mulago hospital respectively
between October 2007-March 2008 (Kulabako et al., 2010).
1.4.4

Cryptosporidiosis
Cryptosporidiosis is a parasitic disease of the mammalian intestinal tract caused by the

protozoa, Cryptosporidium parvum and is mainly transmitted through fecal-oral route in humans.
Studies conducted by Tumwine et al., 2003 at Mulago hospital between November 1999-Januray
2001 revealed that 2,446 children below the age of five tested positive for Cryptosporidium
parvum, of whom 72.7% had acute diarrhea and 191 deaths were also reported during the study.
The prevalence of such a disease in children often leads to death if left untreated.

11

Table 1-2: Waterborne disease burden in Kampala
Disease

Rotavirus Diarrhea

Period of prevalence No.of cases/ Deaths reported

References

Jul 2006-June 2008

615 cases

Mwenda et al.2010

Jul 2006- Dec2012

1844 cases

Odiit et al.2014

Sept 2012- 2013

263 cases

Bwogi et al. 2016

6 deaths
Oct 1995

512 (12% diarrhea)

Ssengooba et al. 1995

Cholera

Oct 2007-Mar 2008

23 cases

Kulabako et al. 2010

Dysentery

Oct 2007-Mar 2008

151 cases

Kulabako et al. 2010

Cryptosporidiosis

Nov 1999-Jan2001

191 deaths

Tumwine et al. 2003

2,446 cases ( 72.7% diarrhea)

Prevention of waterborne diseases can be achieved through improved sanitation and
hygiene practices, improved access to safe drinking water supplies and proper storage of
drinking water. This calls for behavioral change among the people and enforcement of policies/
law by the Government of Uganda.

12

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13

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UN-Habitat. 2007. Situation Analysis of Informal Settlements in Kampala.
WHO and UNICEF, World Health Organization and United Nations Children's
Emergency Fund .2015. Progress on sanitation and drinking-water.
15

CHAPTER 2 - ABUNDANCE OF VIRUSES IN WATERS IN SOUTHWEST
KAMPALA, UGANDA

2.1 Introduction
Adenoviruses, rotaviruses and hepatitis A have been discovered to be prevalent both in
drinking water and raw sewage in slum areas of Kampala (Chung et al., 2013, Katukiza et al.,
2013). The presence of such waterborne viruses has led to the outbreak of illnesses such as
diarrhea and gastro-enteritis with the most vulnerable groups being the elderly and children
under the age of five (Odiit et al., 2014, Mwenda et al., 2010). Though the outbreaks of viral
waterborne related illnesses are not well documented, a few studies conducted in Kampala have
established a correlation between the diarrheal illnesses and occurrence of the viruses in children.
A study conducted by Odiit et al. 2014 revealed 32.3% of children under the age of five admitted
to Mulago Referral Hospital in Kampala, from July 2006 to December 2012 suffered from
diarrhea and vomiting. Mwenda et al., 2010 discovered 39% of the stool samples of children
under the age of five tested positive for rotavirus at Mulago Hospital from July 2006 to June
2008. In addition, 37% of children under the age of five at four hospitals in central Uganda had
severe diarrhea (Bwogi et al.2016). All three studies attributed the diarrhea outbreaks to the
presence of rotavirus in stool specimens.
Many studies conducted in Kampala have focused on the bacteriological characteristics
of water (Byamukama et al., 2000, Fuhrimann et al., 2015, Haruna et al., 2005, Howard et al.,
2003, Muyodi et al., 2009, Kulabako et al., 2007). A few studies have focused on the viral
loading of both water and wastewater (Katukiza et al., 2013, Chung et al., 2013). There was a
need to establish more data on the abundance and distribution of viruses in waters in Kampala.
This was the goal for this study.
16

Rotavirus (RV), hepatitis A (HAV), adenovirus (HAdV) and enteroviruses (EV) were
chosen for this study as they are the most common viruses in wastewater (Katukiza et al., 2013,
Kiulia et al., 2010, Rigotto et al., 2010, Schvoerer et al., 2000, Xagoraraki et al., 2014).
Secondly, the waterborne viruses aforementioned cause various diseases in population groups
such as the elderly and children with low immunity and there is need for virus quantification to
establish the risk associated with their exposure. Rotavirus causes severe diarrhea and is the main
cause of mortality in children under the age of 5 not only in Kampala but also throughout the
world (WHO 2009, Mwenda et al., 2010, Odiit et al., 2014, O'Ryan et al., 2001). Diseases such
as conjunctivitis, gastroenteritis and respiratory diseases are attributed to consumption of food
and water contaminated with HAdV B, C and F (Zou et al., 2012, van Heerden et al., 2005).
Several techniques have been used to detect viruses in environmental samples; cell
culture method, plaque assays and qPCR (Xagoraraki et al., 2014). However, the cell culture
method traditionally used to detect viruses lacks sensitivity, inability to characterize different
viruses and takes a longer time to produce desired results (Lee-Montiel et al., 2011, Moore and
Margolin, 1993). QPCR is a rapid, sensitive and reliable technique for quantifying viruses and it
is efficient at analyzing low concentrations (Martin-Latil et al., 2012, La Rosa et al., 2010). The
amplification process produces results in real time. In this study, we used qPCR method to
quantify waterborne viruses via the DNA and RNA extracted from the environmental samples
obtained from the Bugolobi wastewater treatment plant (WWTP), Nakivubo channel and
Nakivubo swamp. The goal of this study was to establish the abundance of waterborne viruses at
the sampling sites.

17

2.2

Materials and Methods

2.2.1

Sample Collection
A total number of 15 samples from five sampling locations were collected between July

7 and August 8, 2016. Samples were collected biweekly from five locations in Southwest
Kampala at a depth of < 1m; Bugolobi Wastewater Treatment Plant (WWTP) both influent and
effluent, Nakivubo channel (upstream and downstream of the WWTP) and Nakivubo swamp
(Figure 2-1). Virus samples were collected with Argonide cartridges based on EPA’s virus
adsorption-elution (VIRADEL) method. The filter apparatus was sterilized with sodium
hypochlorite and dechlorinated with sodium thiosulphate (2%) to avoid cross contamination and
keep the viruses alive at sampling locations. Argonide cartridges were stored on dry ice and
transported overnight to MSU laboratory for further processing.

Figure 2-1: Sampling locations in Kampala
18

Figure 2-2: Water flow direction across sampling locations
2.2.2

Sample Processing
Sample processing entailed three steps: elution of Argonide cartridge filters, flocculation

and microbial removal.
2.2.2.1

Elution of Argonide Cartridge Filters
Sections of braided tubing from the pressure vessel were attached to the inlet of the

cartridge housing and the inlet port of the pressure vessel was connected to a positive air pressure
source. 1000 mL of prepared beef extract was added into the pressure vessel. The Argonide filter
was placed into the cartridge house, and closed tightly and a 2 L beaker was placed under spout
of cartridge house to collect the filtrate. The air pressure was turned on and off as soon as the
beef extract solution begun to flow until the beef extract filled the cartridge house. The beef
extract made contact with the Argonide filter for 1 minute and the air pressure was turned on
again to let beef extract exit the cartridge house into the 2L beaker. The filter was then removed

19

from the cartridge house and residual beef extract was poured into the properly labeled 2 L
beaker. This was followed by flocculation.
2.2.2.2

Flocculation
The 2L beaker was placed on a magnetic stirrer and stirred at a sufficient speed to

develop a vortex. 1M HCL was added to the beaker until the pH of the beef extract elutes
reached 3.5 +/- 0.1. Beef extract elute was stirred slowly for 30 minutes and after 30 minutes, the
beaker was taken off the magnetic stirrer and contents were poured into the 500 mL centrifuge
bottle to the brim. Bottle was centrifuged at 3,800 rpm for 15 minutes at 4º C and then the
supernatant was discarded by carefully and slowly pouring it out in order not to disrupt the line
of precipitate. 30 mL of 0.15M Na2HPO4 (sodium phosphate) at pH 9.0-9.5 was added to the
bottle and contents were swirled until the precipitate dissolved completely. The contents of 500
mL centrifuge tube were poured into each labeled 30mL centrifuge tube and pH readjusted to
9.0-9.5 with 1M NaOH. The centrifuge tube was centrifuged at 7,500 rpm for 10 minutes at 4º C.
After 10 minutes, the tubes were removed from centrifuge and the supernatant poured carefully
into a new 50 mL centrifuge tube, so as not to disrupt the pellet. The pellet was discarded and the
pH of supernatant adjusted to ~ 7.25 with 1M HCL. The supernatant was further processed for
microbial removal.
2.2.2.3

Microbial Removal
A 0.45 μm filter was placed onto the tip of a 50 mL syringe and the supernatant from 50

mL centrifuge tube was loaded into a 50 mL syringe. The supernatant was forced through the
filter, back into the same 50 mL centrifuge tube. The filter was replaced continually until all the
supernatant was forced through the filter and syringe. The used 0.45 μm filter was removed and
replaced with a 0.22 μm filter on the syringe. The supernatant was loaded back into the syringe
20

and forced through the filter into a clean 50 mL centrifuge tube labeled with the proper number.
This procedure was repeated until all supernatant was forced through. 2000 μL of sample was
then aliquoted from the 50 mL centrifuge tubes, into the cryogenic tubes. The cryogenic tubes
were stored into the -80ºC freezer along with the leftover supernatant. DNA extraction followed
after the flocculation process.
2.2.3

DNA Extraction
Virus DNA was extracted using QIAamp viral RNA mini kit following the protocol in

the manufacturer’s manual. 140µL of the eluted viral samples were used in the extraction step
and 80 µL of viral RNA was obtained. Reverse transcription was then performed to obtain cDNA
using iscript reverse transcription super mix for RT-qPCR (Bio-Rad). Generally, 10 µL of viral
RNA samples, 4 µL of iscript RT super mix and 6 µL of water were used to obtain 20µL reaction
mix for each RT-qPCR reaction. The processed cDNA samples were stored in a freezer at -20oC.
2.2.4

Virus Quantification
qPCR was used to quantify four viruses: adenovirus, rotavirus, enterovirus and hepatitis

A virus. All qPCR analyses were performed in the light cycler with a different cycling program
for each of the viruses (Table 2-1). The primer and probe sequences used during qPCR and RTqPCR were taken from literature (Table 2-2).

21

Table 2-1: QPCR cycling conditions used for detection of targeted viruses
Assays
HAdV
(40,41)

Program

Analysis Mode

Number
of Cycle

Target
(oC)

Hold
(hh:mm:ss)

Denaturation

None

1

95

00:15:00

Amplification

Quantification

45

Cooling

None

1

95
60
72
40

00:00:10
00:00:30
00:00:12
00:00:30

Denaturation
Amplification

None
Quantification

1
45

95
95
55
72

00:15:00
00:00:10
00:00:20
00:00:15

Denaturation
Amplification

Quantification

1
45

95
95
60

00:15:00
00:00:15
00:01:00

Denaturation
Amplification

Quantification

1
445

95
94
60

00:15:00
00:00:20
00:01:00

Xagoraraki et al. 2007

Jothikumar et al. 2005

Hepatitis A

Dierseen et al.2007

Enteroviruses

Rotavirus

Reference

22

Pang et al. 2004

Table 2-2: Primers and probes used for detection of targeted viruses
Target Virus
HAdV (40,41)

Hepatitis A

Enteroviruses

Target Virus
Rotavirus

Primer Name
HAdV-F4041-hex157f
HAdV-F40-hex245r
HAdV-F41-hex246r
HAdV-F4041-hex214rprobe
Forward Primer
Reverse Primer
Probe
EQ-1
EQ-2
EP
Primer and Probe

Sequence (5'-3')
ACCCACGATGTAACCACAGAC
ACTTTGTAAGAGTAGGCGGTTTC
CACTTTGTAAGAATAAGCGGTGTC
FAM-CGACKGGCACGAAKCGCAGCGT-TAMRA
GGTAGGCTACGGGTGAAAC
AACAACTCACCAATATCCGC
FAM-CTTAGGCTAATACTTCTATGAAGAGATGC-BBQ1
ACATGGTGTGAAGAGTCTATTGAGCT
CCAAAGTAGTCGGTTCCGC
FAM-TCCGGCCCCTGAATGCGGCTAAT-TAMRA
Nucleotide sequence (50– 30)

Amplicon
Size
88

Reference
Xagoraraki et al. 2007

89

Jothikumar et al. 2005

141

Dierseen et al. 2007

Location

Rota NVP3-F

ACCATCTACACATGACCCTC

963–982

Rota NVP3-R

GGTCACATAACGCCCC

1,034–1,049

TagMan probe

ATGAGCACAATAGTTAAAAGCTAACACTGTCAA

984–1016

23

Pang et al.2014

2.3 Results
The quantitative PCR revealed that all samples collected from the five sampling locations
were polluted with viruses of interest. The concentrations varied across the sampling locations as
shown in Table 2-3, Figure 2-3 and Figure 2-4. The number of samples that tested positive for
viruses per qPCR runs is shown in Table 2-4.
Table 2-3: Mean genomic copy concentrations of viruses
Sampling Locations

RV

HAV

EV

*1.16×102

7.74×10
(±1.90 ×103)

1.81×103
( ±1.37 ×103)

4.26×10
3
(±2.93×10 )

3

Channel Before WWTP
WWTP Influent

*5.79×101
WWTP Effluent

3

3

5.79×10
3
(±3.09×10 )

3

Channel After WWTP

1.31×103
(± 1.43×103)

6.12×10
3
(±4.83×10 )

Swamp

1.66×102
(±1.04 ×103)

2.73×10
3
(±1.44××10 )

HAdV

6

5.45×105
(±5.22×105)

7

1.17×107
(±6.30×106)

7

9.43×106
(±7.96×106)

6

2.08×106
(±9.65×105)

6

5.72×105
(±3.56×105)

3.09×10
(±9.63×105)
3.91×10
7
(±3.94 ×10 )
1.42×10
(±1.12×107)

4.15×10
6
(±2.51×10 )

3

4.69×10
6
(±5.85×10 )

WWTP - Wastewater Treatment Plant RV-Rotavirus, HAV- Hepatitis A virus, EV- Enterovirus,
HAdV- Adenovirus. Mean values of concentrations of samples that tested positive measured in copies/L. In
brackets is the standard deviation. * No standard deviation.

24

Table 2-4: Number of samples that tested positive per qPCR runs
Rotavirus
Date of
Sampling
07/12/2016

Channel before
WWTP
1/4

WWTP Influent

WWTP Effluent

0/4

0/4

Channel After
WWTP
1/4

07/25/2016

0/4

1/4

1/4

0/6

2/4

08/08/2016

0/6

4/4

0/4

4/4

2/4

Swamp
0/4

Hepatitis A
07/12/2016

4/4

2/6

1/6

2/6

3/6

07/25/2016

3/6

0/6

2/6

0/4

1/4

08/08/2016

0/4

2/6

3/4

1/6

5/6

Enterovirus
07/12/2016

2/2

2/2

2/2

2/2

2/2

07/25/2016

2/2

0/4

1/4

2/2

2/2

08/08/2016

2/2

2/2

0/4

0/4

2/2

Adenovirus
07/12/2016

2/2

2/2

2/2

2/2

2/2

07/25/2016

2/2

2/2

2/2

2/2

2/2

08/08/2016

2/2

2/2

2/2

2/2

2/2

25

a)

b)

Figure 2-3: Distribution of viruses at sampling locations: a) Rotavirus b) Hepatitis A

26

c)

d)

Figure 2-4: Distribution of viruses at sampling locations: c) Enterovirus d) Adenovirus

27

The concentrations of viruses for samples that tested positive ranged from 5.79×101 to
3.91×107 copies/L with RV having the lowest concentration and EV having the highest
concentration.

The one-way ANOVA was used to test the significant difference in mean

concentrations of the viruses at the sampling locations. The lowest concentration of RV was
5.79×101 copies/L in WWTP effluent samples while the maximum was 1.81×103 copies/L in
WWTP influent samples. The genomic copy concentration of RV at the WWTP decreased from
1.81×103 copies/L to 5.79×101 copies/L in influent and effluent samples respectively. There was
no significant difference (p>0.05) observed in RV concentrations across the sampling locations.
In contrast, the genomic copy concentration of HAV at the WWTP slightly increased from
4.26×103 copies/L to 5.79×103 copies/L in influent and effluent samples respectively. Water
samples obtained from the swamp showed the least concentration of HAV of 2.73×103 copies/L.
Similar to RV, there was no significant difference (p>0.05) observed in HAV concentrations.
Among the four viruses of interest, EV concentrations were the most predominant ranging from
3.09×106 to 3.91×107 copies/L. The lowest concentration of EV was 3.09×106 copies/L in
samples obtained downstream from the WWTP. EV concentrations decreased by 2.49 copies/L
at the WWTP, a pattern dissimilar to that observed with the HAV concentrations at the same
location. Similarly, the genomic copy concentrations of HAdV were high, 5.45×105 to 1.17×107
copies/L. The concentration of HAdV increased to 2.08×106 copies/L downstream from the
WWTP after mixing with effluent from the WWTP. This is different from the concentration of
HAV that fluctuated at the same location. HAdV concentrations for WWTP influent and effluent
samples were significantly different (p<0.05) from concentrations observed at the other three
sampling locations. Generally, EV were the predominant enteric viruses followed by AdV, then
HAV and lastly RV.

28

2.4 Discussion
The qPCR method quantified the abundance of enteric viruses in environmental samples.
Enteric viruses such as rotavirus, hepatitis A virus, enterovirus and adenovirus were detected at
the five sampling locations.
Rotavirus concentrations were the lowest compared to the concentrations of the other
three viruses. In contrast, Fumian et al., 2010 evaluated the abundance of RV in raw sewage in
Brazil and revealed that the raw sewage was enriched with RV, ranging from 2.5×10-1 to 1.6×104
genomes per mL. The concentration of RV was higher in treated sewage than in a raw sewage at
the WWTP. The RV trend at the WWTP is comparable to another study conducted at a WWTP
in the Netherlands (Lodder and Husman, 2005). The concentration of RV downstream from
WWTP was higher than the concentration upstream at the same location. The WWTP effluent,
with relatively higher concentration of RV mixed with sewage in the channel hence the increase
in concentration. In addition, industries located along the channel dispose untreated sewage into
the channel. RV is a non enveloped virus, with double stranded RNA surrounded by an inner
capsid outside its core, hence its persistence in the environment (Schwab, 2007) as shown by the
results in this study. However, other factors like UV light, temperature and pH influence
persistence of viruses in the environment. The low concentration of RV in the channel upstream
from WWTP are in agreement with these facts. Temperature influences the denaturization rate of
RNA and protein, hence the higher the temperature the higher the rate of viral capsid destruction
(Gerba, 2007). RV is also less stable compared to HAdV due to its structure.
Hepatitis A virus (HAV) contains a single stranded RNA with capsid resistant to
variability (Pinto and Saiz, 2007). This enables HAV to be resistant to acidic conditions and
temperature changes in the environment (Gerba, 2007). However, the concentrations of HAV
29

were second lowest after concentrations of RV. A study conducted by Rodriguez-Manzano et al.,
2010 revealed similar concentrations of HAV in raw sewage of Barcelona, Spain while no HAV
was detected in raw sewage as revealed by Vantarakis et al., (1999). This could have been due to
non-occurrence of HAV infections at the time of sampling. WWTP effluent samples exhibited
concentrations slightly higher than concentrations of WWTP influent, a pattern dissimilar to that
of AdV at the same location. The low virus reductions of HAdV reveal that the conventional
activated sludge treatment methods at WWTP are ineffective to reduce virus concentrations to
acceptable standards. HAV concentration in channel upstream from WWTP is greater than the
RV concentration due to higher stability of HAV in the environment.
Similarly, enteroviruses (EV) can survive in high temperatures and saline conditions,
making enteroviruses persistent in environmental samples (Gregory et al., 2006, Wetz et
al.,2004). This is evident in the channel upstream from WWTP where the EV concentration,
3.09×106 copies/L was higher than the concentrations of the other three viruses of interest at the
same location. Enteroviruses were the most predominant among the viruses of interest. EV
concentrations are observed to be in the order of magnitude of 106-107. The discharge of WWTP
effluent into the channel increased EV concentration in channel downstream. The Nakivubo
swamp, that acts as a natural filter was observed to have high EV concentrations. The prevalence
of EV in the swamp may be as a result of surrounding industries and homes discharging
untreated sewage into the swamp. Additionally, swamps may act as reservoirs for viruses.
Human adenovirus (HAdV) exhibited high concentrations at all the locations, ranging
from 5.45×105 to 1.17×107 copies/L. These values were higher than 3.16 to 1.38×102 copies/mL
and 7.62 ×10-3 to 7.47 copies/mL detected in other polluted environmental samples (Katukiza et
al., 2013, Haramoto et al., 2010). The detection of HAdV in the channel upstream from WWTP
30

is an indication of the presence of fecal matter. The concentration of HAdV at this location was
the second highest due to the relative stability of virus particles from exposure to high
temperatures. The activated sludge process at the WWTP supported the adsorption of HAdV
onto particles that settled in the clarifier, hence a slight decrease in concentration in WWTP
effluent. An increase in concentration of HAdV in channel downstream from WWTP may be
attributed to mixing with WWTP effluent, which contained high HAdV concentrations. Low
concentrations of HAdV were observed in the swamp. Swamps act as natural strainers (Raphael
et al., 1985) to attenuate pollution in water. The linear double stranded DNA of HAdV
surrounded by a capsid makes HAdV resistant to adverse environmental conditions such as pH
and temperature hence its stability in the environment (Mena and Gerba, 2009, Rigotto et al.,
2011). Furthermore, the intact strand can serve as a template during DNA replication process in
case one strand is destroyed by adverse environmental conditions (Enriquez et al., 1995) .
Generally, the virus concentrations of the effluent samples were found to be comparable
with the concentrations of the influent samples. This implies a low reduction of virus
concentration at the WWTP. The virus reduction of < log 2 at the WWTP was lower than the
log reductions revealed by (Katayama et al., 2008, Hewitt et al., 2011) while comparable to those
observed by (Petrinca et al., 2009). The low virus reduction may be attributed to virus adsorption
onto particles that do not settle in the clarifier and not removed effectively during the
clarification process.
Additionally, several factors influence virus survival in water. High temperatures
denature nucleic acid and protein, consequently destroying the viral capsid (Bosch et al., 2006).
Ultraviolet light causes nucleotides to cross link leading to virus inactivation. PH also influences
virus survival in water. Laboratory processes such as conversion of RNA to cDNA affect
31

quantitative results due to RNA degradation (Fleige et al., 2006).
Bugolobi wastewater treatment plant effluent is discharged into Nakivubo channel, and
then enters the Nakivubo wetland before discharge into Lake Victoria hence the high virus
concentration of up to 4.69×106 copies/L. The high virus concentration can be also attributed to
the untreated sewage released into the wetland from surrounding industries and homes. Natural
wetlands such as Nakivubo swamp are capable of reducing viruses in wastewater through
exposure to sunlight, microbial interactions and plant uptake (Bosch et al., 2006, (Raphael et al.,
1985). However, persistence of viruses in the wetland is a challenge since viruses adsorb
strongly onto soil hence the soil may act as a reservoir for viruses (Powell et al., 2000, Sobsey et
al., 1980, Dowd et al., 1998). Nakivubo swamp discharges water into Lake Victoria, therefore
water pumped from Lake Victoria at the Ggaba water treatment plant requires effective treatment
before distribution and human consumption to prevent waterborne disease outbreaks.

32

2.5 Conclusion and Recommendations
Waterborne diseases caused by viral pathogens remain a major challenge to developing
cities like Kampala. Limited studies have been conducted to establish the distribution and
abundance of enteric viruses in the environment in Kampala. This study established the
abundance of enteric viruses at five locations in Kampala (Nakivubo channel (before and after
WWTP), Bugolobi wastewater treatment plant (Influent and Effluent) and Nakivubo swamp)
during the dry season, from July 7 to August 8, 2016. These data can be used to monitor the
prevalence of diseases such as rotavirus diarrhea, respiratory and gastro-enteritis illnesses among
populations in Kampala. Further studies are recommended to establish the temporal distribution
of viruses at the sampling sites and assess the risk of exposure to waterborne viruses in Kampala.

33

APPENDIX

34

Table A-1: Sample data sheet for fieldwork
SAMPLE DATA SHEET
SAMPLE NUMBER:
DATE OF SAMPLING:
UTILITY NAME:
UTILITY ADDRESS:
COUNTRY:

CITY:

GPS COORDINATES:

SAMPLER’S NAME:
WEATHER CONDITIONS:

WATER pH:
ADJUSTED WATER pH:
FILTER APPARATUS DISINFECTED:

(CHECK) _____ YES _______ NO

FILTER APPARATUS DECHLORINATED: (CHECK) _____ YES ______ NO

INITIAL METER READING:

______________ Gallons

FINAL METER READING:

_______________ Gallons

TOTAL SAMPLE VOLUME:
______________ L
( Final –Initial Meter Readings* 3.7854)

35

Table A-2: Field data sheet for sampling carried out on 07/12/2016

Utility Name

Nakivubo Swamp
Bugolobi Sewage
Plant Effluent
Bugolobi Sewage
Plant Influent
Nakivubo Channel
(After Bugolobi)
Nakivubo Channel
(Before Bugolobi)

GPS Coordinates

Water pH

Initial Meter
Reading
(Gallons)

NKS

459541 E, 32635 N

7.6

379

389.4

39.37

BSPE

456380 E, 35019 N

7.55

411.2

412.7

5.68

BSPI

456168E, 35296 N

7.58

414.7

419.5

18.17

NKCAB

456483 E, 34869N

7.54

391.3

398.4

26.88

NKCBB

456369E , 34939N

7.41

401.1

409.2

30.66

Sample
Number

36

Final Meter
Reading
(Gallons)

Total
Sample
Volume (L)

Table A-3: Field data sheet for sampling carried out on 07/25/2016
Final Meter
Reading
(Gallons)
428.4

Sample
Number
NKS

GPS Coordinates
459541E, 32635 N

Bugolobi Sewage
Plant Effluent

BSPE

456380E, 35019 N

8.32

6.53

436.2

441.3

19.31

Bugolobi Sewage
Plant Influent

BSPI

456168E, 35296 N

8.3

7.07

444

444.7

2.65

Nakivubo Channel
(After Bugolobi)

NKCAB

456483E, 34869N

8.38

7.49

429.9

432.4

9.46

Nakivubo Channel
(Before Bugolobi)

NKCBB

456369E, 34939N

8.12

7.54

432.9

435.8

10.98

Utility Name
Nakivubo Swamp

Water pH
7.95

37

Adjusted
Water pH
7.58

Initial
Meter
Reading
(Gallons)
422

Total Sample
Volume(L)
24.23

Table A-4: Field data sheet for sampling carried out on 08/08/2016
Initial Meter
Reading
(Gallons)
446.3

Final Meter
Reading
(Gallons)
460.4

7.56

475.7

477.2

5.68

8.01

6.51

479

481.4

9.08

456483 E, 34869N

8.07

6.55

461.7

466.6

18.55

456369 E, 34939N

7.96

7.42

467.7

473.9

23.47

Sample
Number
NKS

GPS Coordinates
459541 E, 32635 N

Water pH
7.63

Bugolobi Sewage Plant
Effluent

BSPE

456380 E, 35019 N

7.94

Bugolobi Sewage Plant
Influent

BSPI

456168 E, 35296 N

Nakivubo Channel
(After Bugolobi)

NKCAB

Nakivubo Channel
(Before Bugolobi)

NKCBB

Utility Name
Nakivubo Swamp

38

Adjusted
Water pH
-

Total
Sample
Volume (L)
53.37

Table A-5: Field photos for the research work carried out between 07/12-08/08/2016

Sampling along Nakivubo channel

Sampling at Bugolobi WWTP (Influent)

Effluent culvert

WWTP effluent drains into Nakivubo channel

Sampling point in Nakivubo swamp

Filter apparatus sterilization and dechlorination

Cartridge filter packed with dry ice ready for shipping

39

REFERENCES

40

REFERENCES

Bosch, A., Pinto ́, R. M., & Abad, F. X. 2006. Survival and transport of enteric viruses in the
environment. Viruses in foods, Springer, 151–187.
Byamukama, D., Kansiime, F., Mach, R. L. & Farnleitner, A. H. 2000. Determination of
Escherichia coli contamination with chromocult coliform agar showed a high level of
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