BOVINE REPRODUCTIVE INFECTIOUS DISEASE IN GHANA: PREVALENCE AND
PATHOGENESIS OF EARLY INFECTION WITH AN EMPHASIS ON TRICHOMONIASIS.
by
BENJAMIN ADU-ADDAI

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Pathology
2012

ABSTRACT
FOR DOCTORAL DISSERTATIONS
By
Benjamin Adu-Addai

Reproductive inefficiency is one of the most costly and production limiting problems
facing the livestock development in Africa. Reproductive performance is influenced by a number
of important factors, among which are infectious diseases, which result in lost production.
Research in these diseases is critical in order to overcome these shortfalls.
The objectives of the following studies were to provide a more in-depth epidemiological
analysis of infectious reproductive diseases in Sub-Saharan Africa and further investigate the
cytokine environment and molecular factors involved in the innate immune response of the
bovine reproductive tract to infectious agents which can play such an important role in livestock
production in Africa, in particular Tritrichomonas foetus.
The prevalence of important infectious reproductive diseases in a single herd using
serology was investigated. Significantly, high seroprevalence of infectious bovine rhinotraechitis
and trichomoniasis was demonstrated. The presence of coxiellosis, neosporosis, bovine viral
diarrhea virus, and bovine herpesvirus-4, was demonstrated but not serological evidence of
brucellosis. Significant associations were found between the seroprevalence of the different viral
pathogens but no such association was noted between the other pathogens. The long calving
interval in this herd could be due to the presence of these pathogens. Among the diseases we
identified, we further studied trichomoniasis (T. foetus) and the host immune response in a

mouse model. Serum and reproductive tissues for Th1 and Th2 cytokines (specifically INF-γ,
TNF-α, IL-2, IL-4, and IL-5) via flow cytometry after 5 days of T. foetus infection were
analyzed. Mice of varying ages which had different susceptibility to T. foetus infection were used
to assess whether a Th1 or Th2 cytokine environment was significantly associated with
successful infection. Both Th1 and Th2 cytokines (TNF-α, IL-4 and IL-2) were shown to be
significantly altered in infected animals. Lastly, by using PCR and IHC techniques, the presence
of the novel dual oxidase molecules (DUOX 1 and 2) in the reproductive tract of cows and in
bovine endometrial and trophoblast cell cultures was documented.

However, there was no

significant difference in DUOX 1(P=0.296) or DUOX2 (P=0.480) expression in T. foetus
infected cells versus their respective controls.
In conclusion, several important infectious diseases in Ghanaian cattle which may affect
reproduction, not the least of which is trichomoniasis, were detected.

Further, it was

demonstrated that the early infection has both Th1 and Th2 cytokine involvement and that while
present in the reproductive tract, there is no evidence that the DUOX molecules play a role in
early resistance, but they may be important at other time points.

ACKNOWLEDGEMENTS

I would first like to thank the Almighty God, one above all of us, for granting me the
needed strength and health for this work.
My deepest gratitude is extended to my advisor, Dr. Dalen Agnew, for his excellent
guidance, caring, patience, and providing me with an excellent atmosphere for doing research.
His wisdom and guidance helped to build my scientific creativity and perseverance, for which I
will always be grateful.
I would also like to thank my committee members Dr. Scott Fitzgerald, Dr. John
Kaneene, and Dr. Mohankumar. Their guidance and ideas throughout the years have been
invaluable.
My heartfelt thanks go to Dr. Steve Bolin who accepted me in his laboratory for my first
rotation. I am also grateful to Dr. Charles Mackenzie for his support and often times editing my
work.
My special thanks goes to Dr. Scott Fitzgerald and Dr. Dalen Agnew who in addition to
supervise my dissertation, also supervised my work at the Anatomic Pathology Residency
Program, where I gained a profound depth of knowledge in pathology. I am also grateful to the
entire staff here for their support, cooperation and of course the knowledge they shared with me.
I am also indebted to the Ghana Government for paying my tuition, and Michigan State
University, College of Veterinary Medicine and Graduate Academic Program for funding my
work.

iv

There are certainly many other names that are supposed to be associated with this
achievement because with this numerous assistances made this work a unique one. To all of
such people, big thanks go to you.
Finally, I would like to thank my family. Their continual faith in me and their
love has helped me to overcome any obstacle. My mother, and my uncle for their role in my
education which has brought me this far. My wife, Mavis, and the children, Nana Kofi, Fiifi,
and Abena have provided me with undying love, support, and understanding. May God richly
bless you all.

.

v

TABLE OF CONTENTS

List of Tables

ix

List of Figures

x

Chapter 1

1

Introduction

Chapter 2 Literature Review - Brief introduction to Ghana agriculture
2.1 Agriculture in Ghana
2.2 Livestock in Ghana
2.2.1 Sheep and goats
2.2.2 Pigs
2.2.3 Poultry
2.2.4 Fisheries
2.2.4 Cattle
References
Table

4
5
8
9
9
9
9
10
12
14

Chapter 3 Literature Review - Reproductive Infectious Diseases of cattle in Ghana
3.1 Brucellosis
3.2 Leptospirosis
3.3 Campylobacteriosis
3.4 Infectious bovine rhinotraechitis
3.5 Bovine viral diarrhea
3.6 Trichomoniasis
References

16
16
17
18
19
20
21
22

Chapter 4 Literature Review - Trichomoniasis: a significant disease of cattle in Ghana
4.1 Mechanism of immune response in tritrichomoniasis
4.2 Acquired immunity
4.3 General overview of innate immunity
4.4 Anatomical barriers
4.4.1 Mechanical factors
4.4.2 Chemical factors
4.4.3 Biological factors:
4.5 Humoral barriers
4.5.1 Acute inflammation
4.5.2 Complement system

25
27
29
30
30
30
31
31
32
32
32

vi

TABLE OF CONTENTS

4.5.3 Coagulation system
4.5.4 Iron
4.6 Cellular barriers
4.6.1 Inflammation
4.6.2 Neutrophils and eosinophils
4.6.3 Macrophages
4.6.4 Natural killer (NK)
4.7 Cytokines in innate immunity
4.8 Phagocyte response to infection
4.8.1 Circulating neutrophils/monocytes
4.8.2 Phagocytosis
4.8.3 Respiratory burst
4.8.4 Nitric oxide-dependent
4.9 Non-specific killer cells
4.10 Pathogen determinants
4.11 Tritrichomonas foetus innate immunity
References

32
32
33
33
33
33
33
34
34
34
35
35
37
37
38
38
41

Chapter 5 Field survey of some selected Infectious Reproductive Disease
with emphasis on Tritrichomonas foetus
Abstract
Introduction
5.1 Materials and Methods
5.2 Results
5.3 Discussion
54 Acknowledgements
References
Figures
Chapter 6 The innate immune response in mouse model during acute infection of
Tritrichomonas foetus
Abstract
Introduction
6.1 Materials and Methods
6.2 Results
6.3 Discussion
6.4 Acknowledgements
vii

47
47
48
51
56
57
60
60
64

68
69
69
71
75
77
78

TABLE OF CONTENTS

References
Tables

79
86

Chapter 7 Potentially protective dual oxidase enzymes (Duox1 and Duox2)
in the bovine reproductive tract
Abstract
Introduction
7.1 Materials and Methods
7.2 Results
7.3 Discussion
Conclusion
Acknowledgements
Reference
Figures

95
95
96
99
104
105
108
109
110
114

Chapter 8

125

Conclusions

viii

LIST OF TABLES

2.1: Human population, Cattle population and number of Cattle per 100
persons in Ghana (1984-1996)

14

2.2: The estimated livestock population of Ghana in 2000

15

5.1:

65

ELISA serology results for infectious bovine rhinotraechitis
(IBR), bovine virus diarrhea (BVD), and bovine herpesvirus
4 (BHV-4) from 166 cattle from a herd in Apolonia, in
coastal Ghana.

5.2: ELISA serology results for Q fever (Coxiella burnetti),
brucellosis (Brucella abortus), and neosporosis (Neospora
caninum) from 166 cattle from a herd in Apolonia, coastal
Ghana.

66

5.3: Hemolytic assay results for serum antibody against
Tritrichomonas foetus in 166 cattle from a herd in Apolonia,
coastal Ghana.

67

6.1: Stages of estrus cycle of mice on the 5th day of post infection
with T .foetus

83

6.2: Vaginal homogenate cytokine concentration in T.foetus-infected
and control mice (4 wk and 8wk old) 5 days postinoculation (mean and standard error of the mean (SEM)
values)

85

6.3: Uterine homogenate cytokine concentrations in T.foetusinfected and control mice (4 wk and 8wk old) 5 days postinoculation (mean and standard error of the mean (SEM)
values)

89

6.4: Serum cytokine concentrations in T.foetus-infected and control
mice (4 wk and 8wk old) at 5 days post-inoculation (mean
and standard error of the mean (SEM))

92

ix

LIST OF FIGURES

1.1:

Flow chart, illustrating the order and the procedure of work

3

5.1:

Apolonia the study site, a village close to Accra, located in the
coastal region of Ghana

64

6.1: The percentage of T. foetus infected mice that were successfully

84

infected in either the uterus or the vagina. Note that all 4-wkold mice had vaginal infections and only three uterine after
inoculation. In comparison only 60% (6) of the 8-wk-old mice
had vaginal infections and four uterine.
6.2: The levels of the cytokine TNF-α in vaginal homogenates at 5
days post-infection with T. foetus. A significant difference is
seen between the 8 wk old infected and the 4 wk old infected.
Bar represents SEM. (n=10 in each group), p=0.032.

86

6.3: The levels of the cytokine IL-4 in vaginal homogenates at 5 days
post-infection with T. foetus. A significant difference is seen
between the 4 wk old infected and the 4 wk old sham control
(p=0.0477). Bar represents SEM. (n=10 in each group).

87

6.4: The levels of the cytokine IL-2 in vaginal homogenates at 5 days
post-infection with T. foetus. A significant difference is seen
between the 4 wk old infected and the 4 wk old sham control
(p=0.412). Bar represents SEM. (n=10 in each group).

88

6.5: The levels of the cytokine IL-4 in uterine homogenates at 5 days
post-infection with T. foetus. A significant difference is seen
between the 4 wk old infected and the 4 wk old sham control
(p=0.0428). Bar represents SEM. (n=10 in each group).

90

6.6: The levels of the cytokine IL-2 in uterine homogenates at 5 days
post-infection with T. foetus. A significant difference is seen
between the 4 wk old infected and the 4 wk old sham control (p=
.00063). Bar represents SEM. (n=10 in each group).
x

91

LIST OF FIGURES
6.7: Levels of the cytokine TNF-α in serum 5 days after infection with
T. foetus. There was significant difference between the 4 wk old
infected and the 4 wk old sham control (p=0.0158). Bar represents
SEM. (n=10 in each group)

93

6.8: Tritrichomonas organisms in the lumen of the mouse’s vagina,
and occasionally mixed with the mucinous secretion, as
detected by immunlabelling with anti-T.foetus antibody

94

7.1: Graph showing Raw CT values for the three genes; (A).18SEUk; (B). Bovine Duox - 1B and, (C) Bovine Duox 2B. Note.
The abundance of DUOX 2 in the vagina tissue as compare to
DUOX 1.

114

7.2: Amplification of the three genes in bovine reproductive tissues.
(A).18S (B). Bovine Duox -1 and, (C) Bovine Duox 2.

116

7.3: Graph showing the Delta Ct values of DUOX1 and DUOX2 in
reproductive organs (bars represent the mean CT value from 6
animals with standard deviations). Note. Lower DCT values
indicate higher expression levels.

117

7.4: Localization of DUOX proteins in bovine vagina and uterus by
immunohistochemistry A. Immunostaining for DUOX1 in bovine
vagina. Note the heavy DUOX1 immunostaining in the
cytoplasm of the epithelium. B. Immunostaining
for DUOX
in bovine uterus with light DUOX immunostaining in the
cytoplasm of the epithelium. C. Immunostaining for DUOX2
in bovine vagina. Note the heavy DUOX2 immunostaining
in the cytoplasm of the epithelium. D. Immunostaining for
DUOX2 in bovine uterus There is less DUOX2 immunostaining
in the cytoplasm of the epithelia relative to the other sections.
mmunohistochemistry for DUOX1 and DUOX2, ABC
method; counterstained with Grill 2 hematoxylin. 40x.

118

xi

LIST OF FIGURES

7.5: PCR results of endometrial and trophoblast cells lineshowing
expression levels of 18S, DUOX1, and DUOX2. (A) The varied
expression levels of 18S for each of the cell lines. (B) Note.
The expression of DUOX 1, with amplification of CT values
in the mid 30’s.indicating lower gene expression levels. (C)
Note. No significant expression of either cell.

120

7.6: Localization of DUOX1 protein in bovine endometrial and
trophoblast cultured cells by immunohistochemistry. A.
Immunostaining for DUOX 1 in bovine endometrial cell. Note
the heavy DUOX immunostaining in the cytoplasm of the
epithelium. B. Immunostaining for DUOX1 in bovine
trophoblast. Note The weak DUOX1 immunostaining in the
cytoplasm of the epithelium. Immunohistochemistry for
DUOX1with ABC method; Grill 2 hematoxylin counterstained.
40x.

121

7.7: qPCR amplification of, 18S, DUOX1 (A), and DUOX2 (B) in T.
foetus-infected and uninfected bovine endometrial cells.

122

7.8:

123

DUOX1 (A) and DUOX2 (B) gene expression in cultured bovine
endometrial cells. (Bars indicate standard deviation; data is not
statistically significant).

7.9: Localization of Duox protein in bovine reproductive tissues by
immunohistochemistry. A. Immunostaining for DUOX1 in
cultured T. foetus-infected bovine endometrial cells. Heavy
DUOX1 labeling is present in the cytoplasm of the cells. B.
Immunostaining for DUOX1 with lighter labeling in the
cytoplasm of the uninfected endometrial cells. C.
Immunostaining for DUOX2 in infected bovine endometrial
cells. Heavy DUOX2 labeling is present in the cytoplasm of
the endometrial epithelium. D. Immunostaining for DUOX2
with lighter labeling in the cytoplasm of bovine endometrial
cells. Immunohistochemistry for DUOX1 (1:200) and
DUOX2 (1:25) with ABC method; Grill 2 hematoxylin
counterstain. 40x.
xii

124

Chapter 1
Introduction
Domestic food animals are susceptible to an array of viral, bacterial, protozoan, and
metazoan pathogens which may decrease production efficiency or cause overt disease.
Prevention of diseases is often approached through identification of the causative agent and
appropriate immunologic interventions such as vaccination or immunostimulation. To achieve
this goal, a thorough understanding of the pathogens and the host resistance mechanisms
particularly at the mucosal surfaces such as the gut, respiratory and genital tracts are required.
Sub-Saharan Africa is an excellent example of the economic losses that are possible due
to infectious diseases; it is also an excellent example of the opportunities for intervention if the
disease ecology, the pathogen, and the host are thoroughly understood. In particular,
reproductive losses due to infertility limit the economic advancement of this part of the world,
making the reproductive tract an important focal point for future studies. It is with this in mind,
that the studies described in the following pages, were conducted (Figure 1).
First, the prevalence of important reproductive diseases in Ghanaian cattle was
investigated. It was surprising that only a very few studies have been conducted in this region
and only on a very few diseases. I investigated the presence of important reproductive diseases
in a single herd of cattle, located at Apollonia near Accra in Ghana.
Among the diseases identified in Ghanaian cattle, trichomoniasis, caused by the
protozoan Tritrichomonas foetus (T. foetus) was important. So, secondly using T. foetus as a
model, the host immune response was studied, emphasizing resistance mechanisms which
cooperate to establish protective responses at the mucosal surfaces of the reproductive tract in
vivo at the earliest point of infection, with the goal to stop the initial colonization via the innate

1

immune system responses. Due to the challenges of using cattle as a live animal model, an
established mouse model was used that showed differential infection rates in mice depending on
their ages and an in vitro model of infection using bovine endometrial cell lines was developed.
The cytokine environment in infected mice during the early phases of infection was
investigated, by assaying for Th1 and Th2 cytokines using flow cytometry in 4 and 8 week old
mice, both infected and uninfected. If the cytokine environment can be characterized as to which
is most resistant to initial infection, vaccines can be designed towards this goal.
A novel molecule identified in other mucosal organs as important in innate immunity, the
dual oxidases (DUOX) 1 and 2 was also investigated. First, the presence of this molecule was
documented in the mucosal surface of the reproductive tract in the bovine through PCR and
immunohistochemistry. Their presence was demonstrated and an endometrial cell culture model
of T. foetus infection was developed to look for differences in DUOX1 and DUOX2 expression
between the T. foetus infected endometrial cells and the uninfected cells.
From these studies, I hoped to demonstrate the importance of both field epidemiological
and bench investigations in understanding, and ultimately controlling, infectious reproductive
diseases, with the goal of improving the efficiency of livestock production in Africa and around
the world.

2

PPPPppgsgsgsfsfsf

Prevalence of infectious
reproductive diseases in
Ghana

Survey -Chpt.5
Bacteria
Brucella abortus
Coxiella burnetti

Protozoans
Neospora caninum

Viruses
Infectious bovine rhinotraechitis

Tritrichomonas foetus

Bovine viral diarrhea virus
Bovine herpesvirus4

Pathogenesis of
T.foetus infection

Acquired immunity
immunity
Literature review -Chpt.4

Innate immunity

Cytokine environment during
early infection
In vitro mouse Studies Th1
vs. Th2. - Chpt. 6

Role of DUOX 1 and 2

In vivo and in vitro. PCR
and IHC in cows-Chpt. 7

Conclusion -Chpt.8
cCCChpt.8

Figure1.1. Flow chart, showing the
Figure of research work.
order 1. Flow chart, showing the order
of research work.

Future Directions

For interpretation of the references to color in this and all other figures, the reader is referred
to the electronic version of this dissertation.
3

Chapter 2

Literature Review- Ghana
Brief introduction to Ghana Agriculture
Ghana is located in West Africa. It is bounded in the east by Togo, west by Cote d’Ivoire
and the south by the Gulf of Guinea. It lies within 1 to 11 degrees north of the Equator and is
bisected by the Greenwich meridian.6 The climate is tropical, but temperatures vary with season
and elevation. Except in the north, two rainy seasons occur, from April to July and from
September to November. In the north, the rainy season starts from April to September
(http://www.ghanaweb.com/GhanaHomePage/geography/climate.php; accessed 28 Jun 07).
There are three principal types of vegetation from south to north: coastal savannah, forest zone,
and northern savannah (http://www.africanconservation.org/ghanaprofile.html; accessed 30 Jun
07). The temperature varies between 22C to 33C annual mean, with an equatorial seasonal
variation.6 Many rivers drain the land including the Volta, which has been dammed to form the
largest man- made lake.
Ghana is the first state in the Sub-Saharan Africa to gain political independence from the
British rule in 1957, under the leadership of the late Dr. Kwame Nkrumah, the first president of
the country, who was later overthrown by a military government. Ghana has since changed to a
democratic rule and is considered as one of the leading democratic countries in Africa. The
system of government is a constitutional democracy, which is made up of three arms: the
executive (President, vice President, and 32 elected ministers), the legislature (parliament and its
members), and the judicial (Supreme Court and the chief justice).

4

The total surface area of Ghana is about 239,460 sq. Km. with a population of 20 million,
dominated by the Christian religion (http://www.crawfurd.dk/africa/ghana.htm access 30th June).
It has different ethnic groups, with the following being the major groups and the languages
spoken: the Akan- twi; the Ewe- ewe; the Mole Dagbane- molegabane; the Ga-AdangbeGaAdangbe and the Guan-kyereponi and guan. English is the official language. The Akans are
the major ethnic group occupying mainly from the south to the middle part of the country. From
the southeast to a zone slightly to the north are the Ewes. Between the southeast and the west of
the Accra coastlands are the Ga-Adangbes. The Mole Dangbane occupy the northern and upper
part of the country. The Guans are small groups found in pockets in almost all the other
communities. They are believed to have migrated from other countries to Ghana in search of
jobs in the farming areas.4 Though Ghana has different ethnics groups based on culture and
geographical location, no part in Ghana is ethnically homogenous except perhaps in the
villages. It is worth mentioning that, there is always a high degree of tolerance and
understanding between the various ethnic groups as well as their dependence on God, which may
explain the longstanding peace in Ghana. The country has 10 political administrative regions,
and 110 administrative districts. All regions and districts co-ordinate their activities with the seat
of the government in the capital, Accra.
2.1Agriculture in Ghana
Agriculture plays a central role of the social and economic development of Ghana. The
agricultural sector consists of 5 sub-sectors, which are; crops other than cocoa (63% of
agricultural GDP), cocoa (14%), livestock (7%), fisheries (5%) and forestry (11%).
(http://www.ghanaweb.com: Feature article of Saturday, 4 Feb. 2006 access 30th June 2007).
Presently, the sector employs about 65% of the labor force, accounts for over 40% of the gross

5

domestic product (GDP), contributes about 57% of foreign exchange earnings and provides over
90% of the food needs of Ghana on sustainable basis. (http://www.ghanaweb.com: Feature
article of Saturday, 4 Feb. 2006 access 30th June 2009). This sector also provides raw materials
for the local industries such as soap, food and beverage industries and others.
(http://www.ghanaweb.com: Feature article of Saturday, 4 Feb. 2006 access 30th June 2009)
Agriculture practice in most part of Ghana is not scientifically based. Farmers, the majority of
whom are women, depend on the rudimentary technology for production that always fails to
maximize production.1
It is envisaged that, with the current rate of population growth in Ghana, it is expected to
exceed 36 million by 2020. This means that, there will be pressure for more land acquisition
there by reducing the agriculture land availability per capita from 0.80 hectares (1995) to 0.38
hectares (2020). The immediate solution to this is the government institution of a long term and a
broad based national development policy, Vision 2020. This policy is to rapidly transform
agriculture practices with the aim of meeting the needs of the anticipated population growth as
well as to bring about overall economic growth and its effect on poverty.
(http://www.ghanaweb.com: Feature article of Saturday, 4 Feb. 2009 access 30th June 2010).
This policy more or less will depend on the international community for its achievement.
The peaceful political atmosphere and the stability in Ghana, make it a safe investment
destination in Africa, and therefore it continues to attract more investors. The recent aid to
support its agriculture policy is the amount of $547 million Millennium Challenge Compact
from the US government. Out of this, $240 million shall be used to support the agricultural
program, which is to benefit all types of farmers. This will involve seasonal credit to farmers,
irrigation improvement, and the provision of storage and processing structures. To enhance easy

6

and inexpensive cost of transportation of agricultural products to the consumer market, $143
million shall be used to improve major feeder roads in the farming communities and then $100
million will go into expansion of community services and building of local government capacity
th

(http://www.state.gov/secretary/rm/2006/c18495.htm access 30 June 2007).
The current Accelerated Agricultural Growth Development Strategy (AAGDS), under the
livestock services program, has the goal to promote economic growth and social equity from the
activities of small-scale producers'. Under the new Livestock Development Project, there is to be
an investment more than $40 million over the period 2004-2010 in a number of districts located
in seven regions, focusing mainly on breed development and other husbandry support, animal
health improvement, provision of credit and strengthening of farmers groups and general
capacity building.2 In line with this, Animal Production Department (APD), Veterinary Services
Department (VSD), and the Livestock Planning and Information Unit (LPIU) have been
mandated with specific duties such as: (i) breed improvement, (ii) Forage and pasture
development, (iii) Stock water development, (iv) Disease control, (v) Immunization program,
(vi) vaccine production (e.g., Anthrax spore vaccine), and (vii) continuous education for
farmers.
The animal agricultural development program is supported by a number of research
institutes in Ghana among which are the Animal Research Institute of the Council for Scientific
and Industrial Research (CSIR), and the Animal Science Departments of the three Universities in
Ghana. (Legon, KNUST and, Cape Coast).
Owing to the Accelerated Agricultural Growth Strategy, the government has instituted a
decentralization policy of the Ministry of Food and Agriculture. In this regard, the VSD role is to
assist in the formulation and implementation of projects and program to bring major economic
7

livestock diseases under control as well as encouraging the private sector to play active part in
animal health delivery. This policy has empowered District Veterinary Officers to take
appropriate decisions on disease control and surveillance at the district level and in turn report all
cases to the national Technical Directorate of Veterinary Services. The government
decentralization process and the Accelerated Growth Development Strategy are supposed to
ensure sustainability of livestock program in Ghana.
2.2 Livestock in Ghana
In the previous years, the livestock industry has received less than required attention.
Comparing it to crop sector, there is a lack of sufficient information needed for economic
analysis .9
Table 1 compares the changes in human and cattle population, and number of cattle per
100 people. From the table, there is a consistent declination of number of cattle per 100 persons,
which shows that the growth in human population exceeds that of cattle resource base. The
meaning of this is that there won’t be enough meat for human consumption, and therefore there
will be a need to import meat to supplement. With the current emphasis on animal agricultural
development in Ghana, there is encouragement to revive and increase production. Among the
important categories of animals targeted are: cattle, poultry, pigs and small ruminants (sheep and
goats). A new area of livestock being encouraged is grasscutter (cane rat) and ostrich farming
which is being sponsored by German Technical Cooperation Assistance (GTZ). Most of these,
except for poultry, are reared with rudimentary technology. Approximate populations of
livestock are listed in Table 2.2

8

2.2.1 Sheep and Goats
Every farming household keeps them to supplement the family income. To supplement
the inadequate local supply, there is significant importation from neighboring countries. About
80% of the small ruminant farmers practice small-scale farming, where they employ a semi
intensive system.8
2.2.2 Pigs
In the rural areas, the farmers keep mainly the local breed called Ashanti Black Forest Pig
(ABFP), which are smaller in size and less docile. The commercial farms, which are mainly
located closer to urban areas, keep imported breeds like the Large White, Landrace, Hampshire
etc. Production in the north is limited due to the large Muslim population.
2.2.3 Poultry
Eggs and poultry meat are important components of Ghanaian diets as a source of animal
protein. The poultry sector provides employment for significant number of people in Ghana.
Poultry farms are found in any part of the country. Small, medium, and large-scale producers
form about 60%, 30% and 10% respectively of poultry farmers in the country. Rural poultry
constitute about 80% of Ghana’s poultry population. Poultry production, especially the broiler
industry, is declining as the cost of production in Ghana cannot compete with chicken products
imported from Europe, Brazil, and the United States.8
2.2.4 Fisheries
Fish trading is more popular in the south, where the ocean serves as a major resource. In
the inland sector, fish sources are the rivers and lakes. Per capita consumption is about 20kg
representing 60% of all animal protein. Traditionally, smoking and salting is the mode for
preservation for about 80% of fish caught. Freezing and canning are used mainly for those to be

9

exported. The fish industry supports up to 1.8 million people about 10% of the Ghanaian
population with employment.8
2.2.5 Cattle
The majority of Ghanaians depend on beef for their source of protein. The current
production of beef contributes less than 30% of the beef demanded nationwide.8 Ghana thus
depends on imports to make up its insufficiency. Therefore increasing domestic production is a
goal for making it available at an affordable price for the average Ghanaian.
There are two main breeds of cattle in Ghana known as; (a) Bos taurus and (b) Bos
indicus. The Bos taurus includes West Africa Short Horn (WASH), the most popular breed
constituting about 60% of the total cattle population. 10 The name WASH is a general
descriptive term covering all the variation of small non-humped indigenous cattle. They are
generally black and white in color but sometimes fawn and white. Constitutionally, they are
thick set with fine boned limbs (eg. Ndama, Muturu, etc). The other breed Bos indicus comprise
of white Fulani and Zebu which were originally imported from Northern Nigeria to cross the
WASH for improvement in size and level of milk production. The resulting genotype between
the two is known as Sanga, which has features intermediate between the WASH and the White
Fulani. Cattle production in Ghana is mostly sedentary and agro pastoral, rather than nomadic.
Cattle distribution in Ghana varies from the South to the North. In the North savannah, cattle
herd sizes are generally small, usually between 10 and 50 and may be owned by an individual, a
family, or a village. In the South, herd size are quite large most often 50-1000. These animals
are usually owned by an absentee owner and are cared by hired Fulani herdsmen. In most cases
these owners are businessmen, civil servants, or farmers. The herdsmen are paid monthly or for

10

their remuneration, they are allowed to milk the herd and sell products. In some cases, the Fulani
may also be given the third calf from any cow. 5
The major constraint facing the cattle industry in Ghana is disease. Some of the diseases
of economic importance are: contagious bovine pleuropneumonia, trypanosomiasis, babesiosis,
anaplasmosis, dermatophilosis, and foot and mouth disease.9 Rinderpest had been a significant
disease, but was officially eradicated worldwide in 2010. The role of infectious reproductive
diseases in Ghana, however, has not been well characterized, but as in developed countries,
these likely play a major role. 3, 7

11

REFERENCES

12

References
1. African Development Fund AARDDOCAWR: Multinational Project for Sustainable
Management of Endemic Ruminant Livestock in West Africa (The Gambia, Guinea,
Mali, Senegal). . In: Appraisal Report p. P. 5. 1995
2. Arkoma R: The road to food security and poverty reduction in Ghana. Ghana News
Agency, 2005
3. Bartlett PC, Kirk JH, and Mather EC: Repeated insemination in Michigan HolsteinFriesian cattle: Incidence, descriptive epidemiology and estimated economic impact.
Theriogenology 26: 309-322, 1986
4. Berry LV: Ghana: a country study, 3 ed., p. 382. Federal Research Division, Library of
Congress, Washington, D.C, 1995
5. Hill P: A socio-economic report on cattle ownership and Fulani herdsmen in the
Ashaiman/Dodowa District of the Accra Plains. . 1964
6. Koney EBM, Morrow AN, Heron I, Walker AR, Scott GR. Tick control and
dermatophilosis. International Conference Institute of Tropical Veterinary Medicine
1992; 461-464
7. Lafi SQ: Epidemiological and economic study of the repeat breeder syndrome in
Michigan dairy cattle. I. Epidemiological modeling. Preventive Veterinary Medicine 87
Elsevier Science Publishers B.V., Amsterdam: 98 87, 1992
8. Ministry of Food and Agriculture G: Food and Agriculture Sector Development Policy
pp. 58, 59, 60&61. Delaram Ltd. Accra, Accra, 2002
9. Otchere EO, Okanta. SA: Small holder diary production and marketing in Ghana. , p. 2.
Animal Research Institute, Accra
10.

Otchere EO, Okantah SA: Smallholder dairy production and marketting in Ghana, p. 1.
Accra, Ghana

13

Table 2.1. Human population, Cattle population and number of Cattle per 100 persons in Ghana
(1984-1996)
Year

Humans (x 10)

Cattle head

Cattle per 100
persons

1984

12.3

1.08

8.8

1986

13.04

1.13

8.7

1988

13.74

1.14

8.3

1990

14.47

1.14

7.9

1992

15.24

1.16

7.6

1994

16.02

1.22

7.6

1996

18.0

1.25

6.9

Source: Otchere E.O and Okanta S.A “Smallholder dairy production and marketing".

14

The estimated livestock population in 2000 is indicated in Table 2:
Table 2.2: Livestock Population in Ghana
Animal Species

Total Population

Cattle

1.5 million

Sheep

2.5 million

Goats

3.0 million

Donkeys

14200

Poultry

18 million

Horses

3000

Source: Ghana veterinary services/ministry of agriculture (1996)" Livestock census" 1996

15

Chapter 3

Literature Review- Reproductive Infectious Diseases
Reproductive infectious diseases of cattle in Ghana
Despite the fact that, Ghana has been successful in controlling many serious epidemic
infectious diseases, losses to productivity from other cattle diseases still remain high. These are
some of the most costly and production-limiting problems facing the cattle industry in Ghana,
which contributes to insufficient animal protein present in the average Ghanaian diet. Presently,
only 5% of the average Ghanaian diet is composed of protein of animal origin; the rest is of
carbohydrate sources. 13 While there are likely many inter-related factors involved in the lower
than desired performance of the Ghanaian cattle industry, reproductive diseases are likely to be
significant. Unfortunately, there is little information available on reproductive infectious diseases
of cattle in Ghana.
In other countries where infectious reproductive diseases are well studied, they are
known to create heavy economic losses mainly due to their insidious nature.5, 8, 23 The most
studied reproductive infectious disease pathogen in Ghana is Brucella abortus, and to a lesser
degree, Leptospira spp. 22
3.1 Brucellosis
Brucellosis in Ghana has been extensively studied, partly because of its effect on the
extensive economic losses due to abortion and extended calving intervals and also due to it’s
zoonotic nature. The disease in cattle is usually caused by Brucella abortus, and occasionally by
other species, like B. melitensis, B. suis, B. ovis, and B. canis. Brucellosis is prevalent in many
countries especially in the tropics. The prevalence of brucellosis in Africa has been variably

16

reported from zero to 100% 4, where the wide range of variation was attributed to sample
collection and diagnostic techniques. A survey of cattle in Ghana on the Accra plains indicated
prevalence of 20%-30%, with high rates of abortion and stillbirth.22 The disease can be
transmitted through copulation. In the host, the bacteria are conveyed to the regional lymph
nodes, leading to lymphadenitis. The organism is able to multiply in phagocytic cells like
macrophages 3 and disseminate through systemic circulation to the other organs (spleen, lymph
nodes, uterus, and mammary gland). During pregnancy, the bacteria localize in the uterus and
cause ulceration of the endometrium. Both the membrane and the fetus respond to the infection
by increasing the production of erythritol, a carbohydrate compound, which favors the
multiplication of the bacteria. The bacterial multiplication spreads the lesions to the placental
cotyledons and destroys the villi. Placental insufficiency eventually brings about abortion
(typically during the last trimester), stillbirth, premature birth, or weak calves. Infections are life–
long in the dam. 6
3.2 Leptospirosis
Leptospirosis is a bacterial disease characterized by reproductive failure such as abortion,
stillbirth, reduced milk production, and delivery of weak offspring. It too, is an important
zoonotic disease. The causative organism is Leptospira sp., a spirochaete organism which can be
found in many serotypes. Those serotypes found to be associated with bovine abortion are
Leptospira pomona, L. canocola, L. icterohaemorrhagic, L. grippotyhosa, L. hardjo, and L.
borgpetersenii. 12, 19 Infection is usually through the digestive tract as well as the reproductive
tract, eyes, or skin. Following infection and bacteremia, the organism localizes in the kidneys and
is shed in the urine, for weeks to years 2. Direct or indirect contact with the urine of the infected
animals is major route of infection, though there can be transmucosal transmission via the

17

reproductive, digestive, and respiratory tract. An important factor for occurrence of clinical signs
is the immune status of the herd. In a previously uninfected herd, clinical signs may be seen in all
ages.2
Leptospirosis has been little studied in Africa, except in a few places such as Ethiopia
where Moch 16 described the prevalence of various domestic animals as: 91.2% in horses, 70.7%
in cows, 57.1% in pigs, 47.3% in goats, 43.4% in sheep, 15.4% in camels, and 8.3% in dogs. In
Kenya and Uganda, Ball 1 reported prevalence of 34% in cattle and 9.3% in goat samples.
Twenty Leptospira serovars have been documented from previous studies; 11 of these belong to
the species Leptospira kircheri, eight of which were found in Democratic Republic of Congo
(Zaire); 2 in Kenya and one in Ghana .7 Studies conducted in Tanzania also confirmed the high
prevalence in that country.14 The climatic and socio-economic environment of the African
continent are likely important for the high incidence of the disease; Ghana is not exceptional in
the importance of these conditions.
3.3 Campylobacteriosis
Campylobacteriosis (vibriosis) is a venereal disease of cattle. The species most
commonly involved in the bovine genital tract infection are Campylobacter fetus subspecies
venerealis serotype A, C. fetus subspecies fetus. Serotype A and C. fetus subspecies fetus
serotype B. Among these, infertility in cattle is usually associated with C. f venerealis A (90%)
and C.f.fetus B (10%) and only rarely C.f. fetus A and B 27 The disease prevalence in the tropics
varies: in Malawi, for example, the incidence as reported by Klastrup and Halliwell 18 was 11.5%
in 294 zebu and 11.1% of exotic bulls tested serologically. When the vaginal mucus agglutination
test was performed on the cows, it showed that 53.8% of the herds and 13.4% of the samples
were infected. When the same test was run in Zimbabwe, 33% of the cows sampled were found

18

to be positive10. Chronic infections are common, but acute infections can also be clinically
relevant. Acute infections are associated with infertility and abortion. The chronic phase is
usually associated only with abortion.9

In Ghana, the prevalence of campylobacteriosis in cattle

is not known; however, this does not rule out its presence since management techniques are
similar across these countries, such as natural breeding. Under these conditions, bulls transmit
the infection from one female to another. Spread of the organism to the male is primarily by way
of copulation with an infected female. Bulls can remain carriers for up to 18 weeks after
infection. Older bulls tend to retain the infection permanently in comparison to younger bulls
possibly due to the increase in number and size of the crypts in epithelium of penis.
3.4 Infectious bovine rhinotracheitis
Infectious bovine rhinotraechitis (IBR) is caused by a herpesvirus in the subfamily
Alphaherpesvirinae. The infection is characterized by five clinical forms; respiratory, ocular,
abortion, infectious pustular vulvovaginitis, and encephalitis depending on predilection site of the
individual strain. 25 The disease can be transmitted sexually or by droplet inhalation and the
incubation period is between 4 to 6 days. In venereal form it has been associated with infertility.
In the respiratory form, there is hyperthermia with red nose (inflammation of the muzzle and the
nostril), anorexia and depression. Mortality is rare unless it is complicated with secondary
infections. Infectious vulvovaginitis is typically characterized by a fever and whitish discharge
from the vulva. On examination of the vulva lips, there will be red spots and discrete pustules.25
Inflammation of the prepuce and penis with pustular lesions and preputial exudates are seen in
the males.25 The abortion form is seen when infection is extended directly into the cervix or the
uterus. This is often seen when cows are artificially inseminated with IBR contaminated semen.
The infection of the uterus often affects the cow's estrous cycle and reduces fertility. The uterus

19

can also be infected from systemic spread from a respiratory infection. The disease incidence has
not been reported in Africa; however, the study findings of International Livestock Center of
Africa (ILCA) on Ethiopian indigenous zebu cattle (Tekelye Bekele et al, 1989b) showed more
than 50% prevalence. The fact that Ghana shares similar climatic and socio-economic
environmental factors with Ethiopia suggests that this disease may be significant in Ghana as
well.
3.5 Bovine viral diarrhea
Bovine Viral Disease (BVD) is caused by a virus, belonging to the Pestivirus genus
within the family Flaviviridae. 21 Bovine viral diarrhea (BVD) is one of the most economically
important diseases of cattle that has worldwide distribution and tend to be endemic in most
populations.11 The clinical signs of BVD are often subtle, but the clinical disease syndromes
can be grouped into three categories: acute BVD, in utero infections, and diseases in persistently
infected (PI) animals. 26Acute BVD can vary greatly in presentation from fever, depression, and
runny nose and eyes, to diarrhea and respiratory disease. In utero infections with BVDV can
result in abortion, persistently infected animals, congenital defects, or normal, immuned calves.
20, 24

Persistently infected animals can result from in utero infection as described above, or by

birth from a PI dam. The spread of BVDV infection through a herd is mainly by PI animals;
such animals harbor the virus for life and shed it in high concentration without showing any
immune response. In Africa, a few studies conducted on seroprevalence of the disease have
indicated that the prevalence was varied from 70–83%. 17 In Ghana, there is no research or
published information available on this disease.

20

3.6 Ttrichomoniasis
Trichomoniasis is an insidious, economically damaging venereal disease of cattle with a
worldwide impact. The disease is caused by Tritrichomonas foetus. A report looking at samples
from most south African countries indicated that, out of the 3, 458 samples that were tested
for T. foetus, 142 (approximately 4.1%) were positive. 15 Trichomoniasis has been found to be
related to the form of management practices. No study on tritrichomoniasis in Ghana has been
conducted, though the management practices typical of Ghanaian farmers would likely favor the
spread of this disease. This is the reason for the following study.

21

REFERENCES

22

References
1.

Ball MG: Animal hosts of leptospirosis in Kenya and Uganda. American Journal of
Tropical medicine and hygiene 15: 523-530, 1966

2.

Bogvist S, Cea M, Ibrahim F: Interactions between Brucella melitensis and human
phagocytes: Bacterial surface O-polysaccharide inhibits phagocytosis, bacterial killing,
and subsequent host cell apoptosis. . Infection and Immunity: 2110-2119, 2003

3.

C.C. C: Brucellosis in Africa I&II The prevalence. In: Animal Heath and Production in
Africa, pp. 33:193-198/135:192-198. 1985/1986

4.

Chukwu CC: Brucellosis in Africa I&II The prevalence. In: Animal Health and
Production in Africa. 1985&1987

5.

Clark BL, Dufty JH, Parsonson IM: The effect of Tritrichomonas foetus infection on
calving rates in beef cattle. Australian Veterinary Journal 60: 71-74, 1983

6.

Daoust P-Y: Pathology of the Reproductive System In: Pathology of Domestic Animals,
ed. Maxie M, 5 ed. Saunders Elsevier Publisher. Fifth edition, 2007. Volume 3, 2009

7.

Faine A, Bolin, Perolat Leptospira and Leptospirosis. In: MediSci, ed. 1999-2000 nE.
Melbourne Australia, 1999-2000

8.

Goodger WJ, Skirrow SZ: Epidemiologic and economic analyses of an unusually long
epizootic of trichomoniasis in a large California dairy herd. JAmVetMedAssoc 189: 772776, 1986

9.

Gracia MM, Eaglesome M.D, Rigby C: Campylobacters important in veterinary
medicine. Veterinary bulletin 53 793-818, 1983

10.

J.Terblanche: Bovine vibriosis. Rhodesian Agriculture 76: 43-45, 1979

11.

Kampa J, Alenius S, Emanuelson U, Chanlun A, Aiumlamai S: Bovine herpesvirus type
1 (BHV-1) and bovine viral diarrhoea virus (BVDV) infections in dairy herds: self
clearance and the detection of seroconversions against a new atypical pestivirus. Vet J
182: 223-230, 2009

12.

Lawson JR, Bacterial and mycotic agents associated with abortion and stillbirth in the
domestic animals. Infertility of livestock22-351963

13.

Lenin C: Ghana's agricultural sector and prospects for the future. Institute of Economic
Affairs, Accra, Ghana, 1997

23

14.

Machang'u RS, Mgode GF, Assenga J, Mhamphi G, Weetjens B, Cox C, Verhagen R,
Sondij S, Goris MG, Hartskeerl RA: Serological and molecular characterization of
leptospira serovar Kenya from captive African giant pouched rats (Cricetomys
gambianus) from Morogoro Tanzania. FEMS Immunol Med Microbiol 41: 117-121,
2004

15.

Madoroba E: Prevalence of Campylobacter foetus and Trichomonas foetus among cattle
from Southern Africa. African Journal of Biotechnology Vol. 10. : 10311-10314, 2011

16.

Moch RW, Ebner EE, Barsoum LS, and Botros BA: Leptospirosis in Ethiopia: a
serological survey in domestic and wild animals. J Trop Med Hyg 78: 38-42, 1975

17.

Muvavarirawa P, Mudenga, D., Moyo, D., and Javangwe, Sabater J: Detection of BVD
antibodies in cattle with an ELISA. Onderstepoort Journal of Veterinary Research;
62: 241-244, 1995.

18.

N.O.Klastrup, R.W.Halliwell: Infectious causes of infertility/abortion of cattle in
Malawi. Nordisk Veterinaermedicin 29: 235330, 1977

19.

Naiman BM, Alt D, Bolin CA, Zuerner R, and Baldwin CL: Protective killed Leptospira
borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and
gammadelta T lymphocytes. Infect Immun 69: 7550-7558, 2001

20.

Nicholson F: Is BVD a problem in beef herds in Nova Scotia? NSDAM Newsletter 5:4.

21.

OIE: Terrestrial Manual, Bovine viral diarrhea. 699-711, 2008.

22.

Oppong E: Bovine brucellosis in Southern in Ghana. Bulletin of Epizootic Diseases of
Africa 14: 397-403, 1996

23.

Rae DO: Impact of trichomoniasis on the cow-calf producer's profitability. Journal of the
American Veterinary Medical Association 194: 771-775, 1989

24.

Rice DR, D: Common infectious diseases that cause abortions in cattle. . University of
Nebraska extension bulletin G93-1148-A.,

25.

Searl R: Infectious bovine rhinotraechitis. In: Beef cattle handbook, pp. 1-2. Cattle
producers’ library.

26.

Smith B: Bovine Virus Diarrhea; Mucosal Disease: Large Animal Internal Medicine. ,
pp. 806-814. Mosby Publications, 1996

27.

Vandeplassche, Reproductive efficiency in cattle; a guidline for projects in developing
countries. FAO Animal Production and Health paper, 1982

24

Chapter 4

Literature Review-Bovine Trichomoniasis
Trichomoniasis as a significant disease of cattle in Ghana
Trichomoniasis is an insidious, economically damaging venereal disease of cattle with a
worldwide impact. The disease is caused by Tritrichomonas foetus. In culture, T. foetus is
spindle to teardrop-shaped with a body dimension of 13µm in length and 4 µm in width.
Tritrichomonas foetus has three anterior flagella originating from the basal body and one
recurrent flagellum forming the undulating membrane along the surface of the cell and exiting
through the posterior end. 6, 25 Protruding out of the posterior end is the axostyle, a single ribbon
of highly ordered microtubules spanning the entire length of the parasite. with an undulating
membrane. Tritrichomonas foetus is grouped into the phylum Parabasalia, the order
Trichomonadidia, and into the family Trichomonadidae.
The economic effect of the disease is attributed to the increased days open, reduced calf
crop, reduced average weaning weight because of prolonged conception, culling of infected
animals, and the cost of control and preventive procedures.28, 41, 46 The disease may lead to
metritis, early embryonic death, abortion, pyometra and infertility. 8, 20 Due to the insidious
nature of the disease once the chronic state is achieved, the owners and herdsmen are often
unaware of the presence of the disease in their herds.4 Based on the pathology of the disease and
virulence mechanisms, Trichomonas vaginalis, the causative agent of human trichomoniasis, is
closely related family member to T. foetus. Due to their similarity, T. foetus infection in cattle has
been proposed for use as an animal model to study the human pathogen and disease.21
Trichomoniasis may directly cause the death of the embryo or may do so through uterine
endometritis and marked leucocytic diapedesis into the endometrium.60 While trichomoniasis of
25

cattle has been actively studied in the Americas and to some extent in South Africa, there has
been limited work in sub-Saharan Africa, in particular Ghana.
No studies have been done on it partly due to the complex and sometimes time
consuming nature of diagnosis. Also there is a perception that this disease is not present, or if
present, it has no serious economic consequence and therefore no urgency to study it. This
perception may allow trichomoniasis to spread and exert its economic effect on the cattle
industry. Diseases such as brucellosis, vibriosis, and leptospirosis have been well documented
and may over shadow T. foetus infection. In fact, the possibility of dual infection may be under
appreciated in many clinical cases. However, in Argentina, dual infection rate in ranch bulls was
found to be as high as 11.6% in those areas with extensive cattle raising and natural breeding.16
Similar to Argentina, Asia, and Africa, including Ghana, may also have a high prevalence of
trichomoniasis combine with campylobacteriosis.16 40
Tritrichomonas foetus infection has been found to be related to the form of breeding
management practiced. Prevalence of the disease is higher in those areas, where natural breeding
is practiced.19, 31, 61, 7 The percentage of infection from a study survey is reported to be from 7 to
15.8%.10, 57 Cattle management in Ghana is similar to that of other areas where trichomoniasis is
endemic; i.e., (i) natural mode of breeding, (artificial insemination is rarely used); (ii) breeding
bulls are maintained longer (over 6years); (iii) communal bulls are common; (iv) year round
breeding; and (v) T. foetus infection is not routinely investigated.
The lack of accurate data on the level of the infection in Ghana presents a fundamental challenge
to establishing a control program. As a consequence of this, the disease may continue to
multiply, insidiously and negatively impacting the livestock industry in Ghana.

26

The need to survey trichomoniasis and institute effective management and control
measures has potential human health implication. Human trichomoniasis has attracted more
attention of late, because it is known to predispose to human immunodeficiency (HIV) infection
and cause an abnormal outcome of pregnancy.27, 35 Until recently, trichomonads were thought to
exhibit host specificity, and that T.foetus is associated in nature only with bovine disease such as
vaginitis. However, it has been confirmed that this parasite is causing feline diarrhea.39 In
humans, there have been only two cases of T. foetus like infection. In the first case, this was
manifested in the form of meningoencephalitis in a recipient of allogeneic peripheral blood stem
cell transplantation. 42 The second case was a patient with AIDS, in association with
Pneumocystis pneumonia. 22 The implication is that T. foetus can cross infect human host which
is a threat to veterinarians, farm attendants, abattoir, and laboratory workers from direct contact
with infected animals or animal tissues and potentially to the general public if the mode of
infection is through some other unknown mechanism.
4.1 Mechanism of immune response in tritrichomoniasis
Vaginal and uterine mucosae are portals for sexually transmitted diseases (STD)
including tritrichomoniasis in both humans and animals, the success of which is influenced by
number of factors among which are: extensive layer of mucus, nutrient deficiency, antibody
response, constant flow of vaginal fluid and normal flora as being significant during the
infection. 17 Macrophage and polymorphonuclear cells (PMN) respond to the site of infection as
the first line of defense in a host immune response, which usually results in inflammatory
processes including leukorrhea. 48
Histopathologic studies conducted to examine the changes during early experimental
infections in virgin heifers showed inflammation as predominately occurring after 60 days of

27

infection, 44 with neutrophils and macrophages as the major cellular infiltrates in the
endometrium, stratum compactum, glandular epithelium and the uterine lumen. Following the
recognition of the parasite, immune cells release pro-inflammatory molecules including tumor
necrosis factor- (TNF ), interleukins (IL-1, IL-6, IL-8, IL-12) and nitric oxide. 5 These
molecules aid the recruitment and activation of more immune cells and stimulate hepatic
secretion of acute phase proteins leading to an intense antibody response, mast cell
degranulation, and eventually clearance of the parasite. Leukocyte emigration to the site of
inflammation is aimed at killing the parasites by the action of released enzymes.
In a study using T. vaginalis, a ratio of 10 PMNs to 1 T. vaginalis organism was shown to
kill the parasite by degrading it into pieces and phagocytized it. 48 Neutrophil importance in T.
vaginalis infection has been determined to be dependent on the parasite’s ability to inactivate the
complement involved, through alternate pathway other than that which involves antibody
mechanism for killing the parasite. This was demonstrated in a study during which T. vaginalis
was killed in the absence of parasite-specific antibodies or agglutination; complement from nonimmune sera source was included to serve as an indicator. 48 In contrast, for T. foetus to be
killed by neutrophillic action, parasite-specific antibody and complement are essential. 3
Studies so far have concentrated largely on the acquired immune response to T. foetus
and T. vaginalis. However, the initiation of the innate immune response and the role of early
inflammation in T. foetus infection remain less well understood. Studies conducted so far suggest
that the inflammatory process during early infection with T. vaginalis is stimulated by Toll like
receptors-4 (TLR-4) with an unknown ligand, (possibly lipophosphoglycan (LPG). The TLR-4
is sensitized only by the presence of T. vaginalis. 62 In order to understand and delineate the
whole mechanism involving the innate response to T. foetus and T. vaginalis, more research

28

needs to be done. First a brief overview of what is known about the acquired immune response to
T. foetus and other trichomonads will be followed by a more in depth review of the innate
immune response in general and then more specifically about the innate immune response
against T. foetus.
4.2 Acquired immunity
Specific acquired immunity towards antigens of T. foetus has been well studied. In
particular, the antibody response towards antigens of T. foetus has been reported to act via
complement lysis as well as agglutination and opsonization of the parasites. 13, 29 When animals
are infected naturally, protection and clearance are based on strong vaginal and uterine IgA and
IgG1 production. 58 Vaccination studies have focused on local IgA and IgG1 responses; a
vaccination trial indicated enhanced local IgA and IgG1 responses in addition to high serum
levels of IgG1 and IgG2 when a surface antigen, TF1.17 was used. 9 In another study, it was
noted that, following T. foetus vaccination using a whole cell antigen preparation, there was a
strong uterine IgA antibody response which correlated to faster clearance of T. foetus and less
histological evidence of endometritis. 2 Skirrow and BonDurant 58 reported the clearance of
vaginal infection to be more linked with the increase of the vaginal antibodies (local) than that of
systemic response. The important role of T. foetus membrane antigen in providing protection as
well as curing infected animals through systemic immunization has also been reported .18
Lymphocyte subsets like CD4+/CD8+ T cells have been found to mediate the acquired
immune response in microbial infections. CD4+ T cells respond by up-regulating expression of
interferon (IFN- ). Jovanka M. et al (2001) proved that CD4+ T cells which were induced by a
purified and functionally characterized antigen of T. foetus (so-called Tf190) increased
macrophage activation and consequently suppressed parasite growth. This suggests that Tf190

29

complex (also associated with T. foetus surface lipophosphoglycan (LPG)) contains T cell
epitopes, a similar finding to LPG complex of Leishmania spp. 53
In one experiment, peripheral blood mononuclear cells (PBMC) previously exposed to
purified Tf190, had a phenotypic switch to more CD4+ cells after re-exposure to Tf190. This was
contrary to the case with PBMC exposed to whole T. foetus preparations under the similar
conditions, implying that immunization with Tf190 induces CD4+ T cells which increase during
re-infection of the parasite. Also, it shows that immunizing cattle with Tf190 can prime a specific
subset of CD4+ T cells, including inflammatory T cells and T cells responsible for facilitating
antibody. 1, 11
4.3 General overview of innate immunity
Innate immune response is universal and evolutionarily conserved mechanism of host
defense against infection. Development of the innate response predates the development of the
acquired immune response and it is found in all multi-cellular organisms. It is composed of
receptors and effectors with ancient lineage. Defects in innate immunity are very rare and
almost always lethal. The innate immune system is an evolutionary relic whose major function in
higher vertebrates is to contain the infection until the adaptive immune response sets in. It has
the following components:
4.4 Anatomical barriers
4.4.1 Mechanical factors:
These are mechanical factors like squamous epithelial surfaces which form a physical
barrier that is impermeable to most infectious agents. The skin for example acts as first line of
defense against invading organisms. The desquamation of skin epithelium also helps remove
bacteria and other infectious agents that have adhered to the epithelial surfaces. Movement due

30

to cilia or peristalsis helps to keep air passages and the gastrointestinal tract free from
microorganisms. The flushing action of tears and saliva helps prevent infection of the eyes and
mouth. The trapping effect of mucus that lines the respiratory and gastrointestinal tract helps
protect the lungs and digestive systems from infection. All these same factors are seen in the
female reproductive tract. For example, tight epithelial junctions, mucosal secretions, and
myometrial contractions are important factors in preventing microbial access to tissues in cow.
4.4.2 Chemical factors:
Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase found in
tears, saliva and nasal secretions can breakdown the cell wall of bacteria and destabilize bacterial
membranes. The low pH of sweat and gastric secretions prevents growth of bacteria. Defensins
(low molecular weight proteins) found in the lung and gastrointestinal tract have antimicrobial
activity. Surfactants in the lung can act as opsonins (substances that promote phagocytosis of
particles by phagocytic cells). In the reproductive tract of cow, vaginal commensals such as
Lactobacillus spp. produce lactic acid and hydrogen peroxide to create a low (< 5) pH
environment. This helps to protect the lower reproductive tract from pathogenic micro-organisms
and thereby reduces the possibility of infection reaching the upper reproductive tract. 45 In
addition to this, there are defensins which have antimicrobial properties.
4.4. 3 Biological factors:
The normal flora of the skin and the gastrointestinal tract can prevent the colonization of
pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for
nutrients or attachment to cell surfaces. Normal flora has also been shown to be critical in the
protection of the reproductive tract, in particular, the vagina. 45

31

4.5 Humoral barriers
4.5.1 Acute inflammation:
Humoral factors, here defined as blood-borne factors, including but not limited to
antibodies, which play an important role in inflammation, are involved early in edema formation
and the recruitment of phagocytic cells. These humoral factors are found in serum or they are
formed at the site of infection. Only later are antibodies produced.
4.5. 2 Complement system:
This forms a major humoral defense mechanism which in the early immune response is
non-specific. Complement activation early in the immune response can lead to increased
vascular permeability, recruitment of phagocytic cells, and lysis and opsonization of bacteria
(considered the “alternate” complement system). Later, during or after the acquired immune
response takes hold, complement interacts with antibodies to provide very specific killing
functions. Complement has been shown within the vaginal lumen and likely plays an important
role in the control of pathogens, particularly T. foetus. 32
4.5.3 Coagulation system:
Some products of the coagulation system can contribute to the non-specific defense of a
mucosal barrier due to their ability to increase vascular permeability and act as chemotactic
agents for phagocytic cells. Also some of the products of the coagulation system can have nonspecific antimicrobial functions. For example, beta-lysine protein produced by platelets during
coagulation can lyse many Gram-positive bacteria by acting as a cationic detergent.
4.5.4 Iron:
Iron serves as an essential nutrient for bacteria. By binding to and sequestering iron,
lactoferrin and transferrin limit bacterial growth. The interaction at the mucosal level with iron,

32

however, can be complicated, as T. vaginalis has been shown to penetrate mucin layers more
effectively in an iron deficient environment.37
4.6 Cellular barriers
4.6.1 Inflammation:
One early role of the inflammatory response is recruitment of neutrophils, eosinophils,
and macrophages to sites of infection as the main line of defense in the non-specific innate
immune system.
4.6.2 Neutrophils and eosinophils:
Neutrophils are recruited to the site of infection where they phagocytose invading
organisms and kill them intracellularly. In addition, neutrophils can lead to collateral tissue
damage to other pathogens and adjacent healthy tissue, sometimes compounding the problem.
Eosinophils, more commonly associated with allergic diseases, hypersensitivity, or parasite
infections have proteins in granules that are also effective in killing certain parasites.
4.6.3 Macrophages:
Tissue macrophages and newly recruited monocytes, which differentiate into
macrophages, also function in phagocytosis and intracellular killing of microorganisms. In
addition, macrophages are capable of extracellular killing of infected or altered self target cells.
Macrophages also can contribute to tissue repair and act as antigen-presenting cells, required for
the induction of specific immune responses.
4.6.4 Natural killer (NK) and lymphokine activated killer cells:
These cells can nonspecifically kill virus infected and tumor cells. They are not part of
the inflammatory response but they are important in nonspecific immunity to viral infections and

33

tumor surveillance. There is some debate as to whether these cells exist at all in cows, but
granular lymphocytes may play a similar role.36
4.7 Cytokines in innate immunity
Cytokines are the hormonal messengers responsible for most of the biological effects in
the immune system, such as cell mediated immunity and allergic type responses. The effect of
cytokines depends on the balance of Th1 and Th2 pathways based on the “T-helper” cells
involved in the process. In mice, Th1 cytokines is typically IFN-γ, while Th2 cytokines include
IL-2, IL-4, IL-5, and IL-8. Some cytokines, like TNF-α can induce inflammation in either
milieu.
The Th1/Th2 pathway paradigm in bovine system is not as well defined as it is in other
species. Sometimes this response seems to be heterogeneous, inducing both Th1- and Th2-like
responses, whereas in other examples predominant Th1 or Th2 responses occur. 12 For example,
T-helper responses induced in Th clones against the rhoptry-associated protein-1 of Babesia
bigemina are biased toward Th1, as characterized by a strong IFN-γ response and a weak IL-4
response.

51

In contrast, clonal T helper responses to Fasciola hepatica-infected cattle were

characterized by strong IL-4 responses and weak IFN-γ. 12
4.8 Phagocyte response to infection
4.8.1 Circulating neutrophils and monocytes:
These cells respond to antigen signals generated at the site of an infection. Such signals
include N-formyl-methionine containing peptides released by bacteria, clotting system peptides,
complement products and cytokines released from tissue macrophages that have encountered
bacteria in tissue. Most of these cells stimulate endothelial cells near the site of the infection to
express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the

34

surface of phagocytic cells and cause the phagocytes to adhere to the endothelium. Vasodilators
produced at the site of infection cause the junctions between endothelial cells to loosen and the
phagocytes then cross the endothelial barrier. Once in the tissue spaces some of the signals
attract phagocytes to the infection site by chemotaxis. The signals also activate the phagocytes,
which results in increased phagocytosis and intracellular killing of the invading organisms.
4.8.2 Phagocytosis:
Phagocytic cells have a variety of receptors on their cell membranes to which infectious
agents bind to these cells. These include Fc receptors, complement receptors, scavenger
receptors, and toll-like receptors. After attachment to a bacterium, the phagocyte engulfs the
bacteria and the bacterium is enclosed in a phagosome. During the process, the granules or
lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a
bacterium engulfed in a phagolysosome which contains the contents of the granules or
lysosomes.
4.8.3 Respiratory burst:
The process of phagocytosis is accompanied by an increase in glucose and oxygen
consumption, referred to as the “respiratory burst.” The respiratory burst results in the
production of number of oxygen-containing compounds which kill the organism being
phagocytosed. The respiratory burst can take on different forms:
Oxygen-dependent, myeloperoxidase (MPO)-independent intracellular killing: During
this process, glucose is metabolized via the pentose monophosphate shunt and NADPH is
formed. Cytochrome B, which was part of the granule, combines with the plasma
membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen
to oxidize the NADPH. The result is the production of superoxide anion. Some of the

35

superoxide anion is converted to H O and singlet oxygen by superoxide dismutase. 23, 47,
2

52

2

In addition, superoxide anion can react with H O resulting in the formation of
2

2

hydroxyl radical and more singlet oxygen. The result of all of these reactions is the
-

production of the toxic oxygen compounds superoxide anion (O ), H O , singlet oxygen
2

2

2

1

( O ) and hydroxyl radical (OH•).
2

Oxygen-dependent, MPO-dependent intracellular killing: In this type, the azurophilic
granules fuse with the phagosome and myeloperoxidase is released into the
-

phagolysosome. MPO utilizes H O and halide ions (usually Cl ) to produce hypochlorite,
2

2

a highly toxic substance. 24,30,33,34 Some of the hypochlorite can spontaneously break
down to yield singlet oxygen. The result of these reactions is the production of toxic
-

1

hypochlorite (OCl ) and singlet oxygen ( O2).
Detoxification reactions: neutrophils and macrophages have means to protect themselves
from the toxic oxygen intermediates. These reactions involve the dismutation of
superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of
hydrogen peroxide to water by catalase. 30
Oxygen-independent intracellular killing: In addition to the oxygen-dependent
mechanisms of killing there are also oxygen–independent killing mechanisms in
phagocytes. 23, 52 Cationic proteins (cathepsin) released into the phagolysosome can
damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin
chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break
down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent
killing pathways are able to kill bacteria. However, since the oxygen-dependent
36

mechanisms are much more efficient in killing, patients with defects in these pathways
are more susceptible and get more serious infections.
4.8.4 Nitric oxide-dependent:
Binding of bacteria to macrophages, particularly binding via Toll-like receptors (see below),
results in the production of TNF-alpha, which acts in an autocrine manner to induce the
expression of the inducible nitric oxide synthetase gene (i-nos) resulting in the production of
nitric oxide (NO). If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric
oxide will be produced. Nitric oxide released by the cell is toxic and can kill microorganisms in
the vicinity of the macrophage.26
4.9 Non-specific killer cells
They include several cells like NK cells, activated macrophages, eosinophils, and mast
cells. These cells are capable of killing foreign and altered self target cells in a non-specific
manner.
In the innate response to virus infection and altered self (transformed cells) NK cells utilize two
kinds of receptors on their surface: NK receptor and inhibitory receptor. When the NK receptor
encounters its ligand on a target cell, the NK cell is signaled to kill. However, if the inhibitory
receptor also binds its ligand (MHC class I) then the killing signal is repressed. Normal cells
constitutively express MHC class I on their surface; however, virus infected and transformed
cells down regulate expression of MHC class I. Thus, NK cells selectively kill virus-infected and
transformed cells while sparing normal cells. Innate responses to extracellular microorganisms
(parasites) also include eosinophils, a specialized group of cells with the ability to engage and
damage large extracellular parasites such as schistosomes. Activated eosinophils release their
granule components including major basic protein, eosinophil peroxidase (a cationic

37

hemoprotein), and eosinophil cationic protein (a ribonuclease that is an eosinophil-specific toxin
that is very potent at killing many parasites).
4.10 Pathogen determinants recognized by the innate immune response
These are determinants recognized by components of the innate (non-specific) immune
system which differ from those recognized by the adaptive (specific) immune system. Antibodies
and B and T cell receptors recognize discrete determinants and demonstrate a high degree of
specificity, enabling the adaptive immune system to recognize and react to a particular pathogen.
In contrast, components of the innate immune system recognize broad molecular patterns found
in pathogens but not in the host. Thus, they lack the high degree of specificity seen in the
adaptive immune system. The broad molecular patterns recognized by the innate immune system
have been called PAMPS (pathogen-associated molecular patterns) and the receptors for PAMPS
are called PRRs (pattern recognition receptors). A particular PRR can recognize a molecular
pattern that may be present on a number of different pathogens enabling the receptor to
recognize a variety of different pathogens.
4.11 Tritrichomonas foetus innate immunity
Vaginal and uterine mucosa is the portal entry for sexually transmitted diseases (STD)
like trichomoniasis. Tritrichomonas foetus is an extracellular organism and infection in cows is
generally self-limiting; because of this, animal health researchers have focused on the role of
acquired humoral immune responses on resistance rather than the acquired immunity responsible
for protection against T. foetus. Host and parasite-mediated factors likely influence the initial
colonization, development of disease, and the ultimate outcome of infection. For successful
colonization in the female reproductive tract, T. foetus requires secreted proteases14, 38, 59 and
adhesion to the surface of the host cell.38 Glycoconjugates on the surface of T. foetus, such as

38

lipophosphoglycan (TF-LPG), facilitate parasite adhesion to bovine vaginal epithelial cells,56
with subsequent contact-dependent parasite-mediated cytotoxicity. 15
In a study examining histopathologic changes during early infection in virgin heifers,
inflammation was said to occur usually after 60 days of infection. 44 In this study, predominant
cellular infiltrates were found to be neutrophils and macrophages within the endometrium,
stratum compactum, glandular epithelium, and the uterine lumen. In related studies that
examined aborted fetuses from infected cattle, it was noted that macrophages and neutrophils
played an important role during infection.49, 50 In this study, tissues where parasites were found,
were usually associated with heavy neutrophil and macrophage infiltration. Also, phagocytized
trichomonads were seen within macrophages and giant cells. In a study of neutrophil killing
mechanism of T. foetus and T. vaginalis, it was noted that neutrophils kills T. vaginalis without
parasite-specific antibodies or agglutination, however, complement derived from non-immune
sera was required.48 On the other hand, neutrophil killing of T. foetus was noted to be most
effective when parasite-specific antibody and complement were present.3 In other studies, nitric
oxide was shown to be an effector of macrophage-mediated cytotoxicity towards the human
pathogen T. vaginalis.43 This study was the first to demonstrate reactive nitrogen species (RNS)
as were able to kill the extracellular venereal parasites; though, this did not implicate RNS as
being critical in the prevention of the establishment of trichomonad infection within the
reproductive tract.
Mechanisms that initiate innate responses and inflammation during T. foetus infection are
currently unknown. Studies using the human pathogen T. vaginalis, indicated that inflammation
during infection is cause by stimulation of Toll-like receptor 4 (TLR-4) with unknown
substances present only during infection with T. vaginalis.62This study did not directly implicate

39

T. vaginalis/TLR-4 engagement; however, it pointed to a mechanism whereby inflammation is
induced. Related studies with T. vaginalis have demonstrated that the parasite has the ability to
induce IL-8 production from both human monocytes 55 and human neutrophils.54 Studies with T.
vaginalis indicate there are most likely similar mechanisms whereby T. foetus is also able to
initiate inflammation within the bovine host. Thus, factors such as proteases and TF-LPG, which
facilitate parasite colonization, are directly affected by the host immune response.
Since most studies have been focusing on acquired immune responses in trichomoniasis,
information such as how the innate response affects early colonization and establishment of
infection in vivo is not available. Understanding the mechanism of effective early immune
response could curtail T. foetus infection and other similar resultant pathogens.

40

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46

Chapter 5

Field Survey of Some Selected Infectious Reproductive Diseases in Ghanaian Cattle
Abstract
Infectious reproductive diseases of cattle can severely impact the economic viability of
the African rural sector. Despite this importance, relatively little is known about the prevalence
of reproductive pathogens in African cattle. To emphasize the importance of these diseases, we
carried out a cross-sectional study on a cattle herd with decreased fertility (calving interval of 2.5
years; instead of 1.15years under improved conditions for evidence of seven infectious
reproductive diseases: bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis
(IBR), bovine herpesvirus-4 (BHV-4), Tritrichomonas foetus (Tf), Coxiella burnetii (Cb),
Neospora caninum (Nc), and Brucella abortus (Ba). The herd was composed of over 700 cattle
from which 18 bulls and 148 cows (2 to 15 years old) were tested. Serum antibodies (ELISA)
indicated exposure to IBR (69.0%), BVDV (13.0%), BHV-4 (20.6%), Cb (6.1%), Nc (17.7%),
and Ba (0%), and a hemolytic antibody assay detected Tf in 48% of these 166 animals. A
significant association was found between all the viral pathogens but not between the other
pathogens. This study, the first in Ghana looking at a range of infectious agents, underscores the
importance of reproductive infections as potentially limiting factors in livestock production in
Africa.

47

Introduction
Conditions that interfere with reproduction in cattle are of great economic importance to
the rural sector of developing countries, particularly those in Africa. The reproductive rate, as
measured by the calving interval, is very often lower in African cattle than in those of more
developed countries. 16, 23This represents a major economic loss for the farmer and the country as
a whole. Although infectious conditions, including viruses, bacteria and protozoa, are likely to be
major contributors to poor calving intervals in Africa, relatively few studies have been carried
out across this vast and rurally oriented continent. To emphasize the need for more information
on the prevalence and role of infectious conditions of the reproductive tract in Africa, we have
examined the profile of a typical West Africa cattle herd in Ghana. This is the first simultaneous
study of multiple infectious agents in Ghanaian cattle.
A range of infectious agents can reduce the calving rate of cattle by causing early
embryonic loss, abortion, multiple returns to estrus, and general infertility. Associated with these
reproductive losses are decreases in milk production, untimely weaning and poor weight gain in
calves, all of which can result in considerable economic loss through reductions in the overall
productivity of livestock. In countries where infectious reproductive diseases have been more
thoroughly studied, they are known to create heavy economic losses, in part due to their
insidious nature. 7, 12, 27, 29
Viral reproductive diseases are likely to be of importance in Ghana. Infectious bovine
rhinotraechitis (IBR), a herpes virus infection, can affect a wide range of tissues including the
reproductive tract where it can cause vulvo-vaginitis, retained placenta, and abortion. 22 Infection
of the reproductive tract occurs from either systemic spread of a respiratory infection or from
direct inoculation of the cervix or the uterus during artificially insemination with contaminated

48

semen or during breeding with an infected bull. Bovine herpesvirus-4 (BVH-4) is known to
induce a postpartum metritis and pelvic inflammatory disease and is likely to contribute to longterm reproductive tract problems. 15 BVDV affects domestic and wild ruminant species
worldwide with a serological prevalence often more than 60%.14 18 32 Bovine viral diarrhea virus
can lead to abortion, stillbirths, congenital defects, and neonatal death, as well as the more
widely recognized immunocompromising and diarrheal diseases of older cattle.14
Bacteria are also significant causes of reproductive tract infection. Coxiella burnetii, an
intracellular pathogen causing Q fever, is an important reproductive infectious agent distributed
worldwide with the exception of New Zealand .2, 28, 34 The disease, typically spread via the
products of abortion, affects all domesticated ruminants, primarily goats and sheep, causing
reproductive failures such as abortions, stillbirth, infertility, and the weakening of offspring.
Animals may show no symptoms and can remain chronically infected.3 28 Brucellosis is
prevalent in many countries, and in Africa, the incidence was reported to vary from nearly zero
to 100%,

6, 24

with the wide variation attributed to differences in sample collection and

diagnostic techniques used. This disease in cattle is usually caused by Brucella abortus and
occasionally by other species, B. melitensis, B. suis, B. ovis, and B. canis. Leptospirosis and
campylobacteriosis also can contribute to reduced reproductive tract function in cattle.
Neospora caninum is a protozoan that can affect cattle throughout the world causing
significant economic loss through abortion especially in countries with intensive dairy
production .9 35 Although this disease can cause abortion at any stage of pregnancy, infected
cows can also give birth to either healthy or diseased, sub-clinically infected calves.9 Another
protozoan important to bovine reproductive diseases is Tritrichomonas foetus which causes
irregular breeding cycles, repeat breeding and longer calving intervals as well as early pregnancy

49

loss and occasionally pyometra. Trichomoniasis has a worldwide distribution and is a major
cause of infertility in naturally bred cattle in many countries. 1, 12, 13, 26 Toxoplasmosis also is
considered an infection of the reproductive tract of cattle but is thought not to be of great
significance.8
The rural sector in Ghana is typical of many African countries where infectious diseases
represent one of the most costly and production-limiting problems facing the cattle industry.
Losses in productivity from disease in cattle are a problem contributing to malnutrition and the
excessive reliance on livestock importation. The current cattle production in Ghana satisfies less
than 30% of the national beef demand and imports are needed to make up for this insufficiency.
11

The most studied reproductive infectious disease in Ghana is brucellosis and most, if not all, of

the other potentially economically important reproductive infections listed above have not yet
been studied in Ghana. A survey of cattle in Ghana on the Accra plains in the 1960’s indicated
an incidence of brucellosis at 20%-30%, with high rates of associated abortion and stillbirth .25
The lack of data on the other infectious reproductive diseases of cattle in Ghana presents a
fundamental challenge to establishing and offering effective veterinary management programs to
increase cattle production. A range of infectious diseases is likely to be present because the cattle
management practices in Ghana, such as natural breeding, the use of aged bulls (over 4 years) in
communal settings, year round breeding, and the lack of routine veterinary care, are known in
other developing countries to be associated with a high prevalence of these reproductive tract
diseases 1, 13, 26
This present report examines a typical cattle herd, with reduced fertility, to determine
the range of reproductive tract infections present by using a cross-sectional study design to detect
the presence of seven important reproductive tract infectious agents (Chapter 1; figure 1),

50

addressing the hypothesis that multiple reproductive pathogens are present in Ghanaian cattle.
This cattle herd is typical of those seen in West Africa and managed by Fulani cattlemen, and
using breeding practices that are commonly to such mobile herds in Africa.
5.1 Materials and methods
5.1.1 Study area and herd description
The study was conducted in Apolonia, a village close to the capital city, Accra, located in
the coastal region of Ghana in West Africa (Chapter 5; Figure 5.1). The climate is tropical, but
temperatures vary with season and elevation. Two rainy seasons occur, from April to July and
from September to November. The daily mean temperature varies between 22 C and 33 C, with
an equatorial seasonal variation. The selected herd, sampled between July and August 2009, was
composed of approximately 700 adult mixed breed African Sanga cattle owned by multiple
owners and managed by Fulani herdsmen. Herdsmen were queried for the current age and
calving history of each cow as well as the routine husbandry procedures practiced. The study
subjects have not been previously vaccinated against any of the diseases investigated. At the time
of surveying, only 2.4% of the cows were found to be pregnant. At night cattle were kept in
small paddocks/kraals near the herdsmen’s homesteads. Milking was done, twice a day, at about
06:00 am and 03:00 pm each day with calves being allowed to suckle for a few minutes before
and after milking. Animals were let out for grazing twice a day after each milking.
5.1.2 Sampling.
Blood was collected by jugular venipuncture from 166 animals (148 of approximately 670
cows and 18 of approximately 30 bulls taken proportionately from each kraal). The minimum
sample size was calculated with a power of 0.8 and using the lowest assumed prevalence of any
of the diseases of interest (approximately 10% as predicted from the literature); i.e.,

51

approximately 116 animals (Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source
Epidemiologic Statistics for Health, Version 2.3.1. www.OpenEpi.com, updated 2011/23/06,
accessed 2012/04/12). Cattle were selected by stratified random procedure, proportional to the
size from each kraal. At the end of each day, the blood samples were centrifuged and about
1.8mls of serum were collected into cryovials and stored at 4oC until they were used within 1
week. Commercially available serum ELISA tests was used to determine exposure to the
following pathogens: infectious bovine rhinotraechitis virus (IBR), bovine viral diarrhea virus
(BVDV), bovine herpes virus 4 (BHV-4), Neospora caninum, Coxiella burnetii, and Brucella
abortus (see below for assay details). A hemolytic assay was used to detect antibodies to T.
foetus.4 Vaginal and preputial fluid washes were inoculated into a commercial test kit for
isolation of T. foetus.a Each serum sample in the ELISA and hemolytic assays was tested in
replicate and the value reported was an average of the two.
5.1.3 Infectious bovine rhinotraechitis (IBR), bovine viral diarrhea virus (BVDV), bovine
herpesvirus 4 (BHV-4) ELISA
Antibodies to IBR, BVDV, and BHV-4 in sera were detected using an indirect commercial
ELISA kit, b according to the manufacturer’s instructions. Briefly, all reagents were brought to
room temperature at least 30 minutes before starting the test. Ten microliters of the serum
samples and positive controls were diluted 1:100, shaken briefly, and 100-μl aliquots of the
dilute samples (1:100) were added to the test wells. The microplates were incubated at 37°C for
one hour, with gentle agitation after 30 min. Wells were rinsed with washing solution and each
plate was emptied by flipping it over sharply above a sink followed by tapping the plate upside
down against a piece of clean absorbent paper to remove all the liquid. This was repeated three
times. The conjugate was diluted 1:50 in the buffer and 100 μl of the dilute conjugate solution

52

was added to each well. The plates were incubated for 1 hour at 37°C, and washed as previously
described. Chromogen diluted in substrate solution was applied to the plate immediately in
volumes of 100 μl per microwell. The plates were incubated for 10 minutes at room temperature
and 50 μl of stop solution per microwell was added. Optical densities in the microwells were
read using a plate reader and a 450 nm filter immediately after stopping.
The results of the test were then expressed as percentage with the following formula:
S/P% = ((ODsample – ODnegative control)/ (ODpositive control – ODnegative control)) x 100. To
overcome the fact that only one serum sample was examined from each cow (not paired sera),
the commercial assay was validated against serum samples with a known virus neutralization
(VN) titer and a conservative threshold determined. Nineteen serum samples with VN titers
varying from < 4 to 512 for IBR and < 4 to 2048 for BVDV were assayed as described above.
The S/P% results varied from 2.5% to 120% for IBR and 0.3 to 88% for BVDV (specific data
not shown). The correlations between the tests for IBR and BVD were found to be r=0.4776
(p=0.0386), 95% CI (0.029, 0.765) and r=0.7558 (p=0.0002), 95% CI (0.459, 0.900)
respectively. Based on these correlations, a cutoff value for a positive IBR was set at S/P% ≥
75% and that for a positive BVD was set at S/P% ≥ 50%. No VN assay for additional validation
of the BHV-4 was available, so positive results were conservatively interpreted as S/P% values
corresponding to values equal or greater than the positive control.
5.1.4 Brucella abortus and Q fever ELISA
Brucella abortus and Q fever (C. burnetii) antibodies were detected using indirect
commercial ELISA kits according to manufacturer’s protocol.

c, d

For B. abortus, serum samples

were diluted at 1:10 and for Q fever, serum was diluted 1:400. The absorbance (OD) was
measured at 450 nm by spectrophotometer. The results of the test were then expressed as
53

percentage using the following formula: S/P% = ((ODsample – ODnegative control)/ (ODpositive
control –

ODnegative control)) x 100. Serum samples for B. abortus with the S/P% < 80% was

considered as a serum from an animal which has not had exposure to B. abortus, while serum
sample with S/P% ≥ 80 was classified as positive. Serum samples for Q fever with SP% < 30%
were classified as negative, samples with SP% ≥ 30% but < 40% was classified as suspect and
samples with S/P% ≥ 40% were classified as positive per the manufacturer’s instructions.
5.1.5 Neospora caninum ELISA
Neospora caninum antibodies were detected using indirect ELISA according to
manufacturer’s protocol.e For N. caninum serum samples were diluted at 1:100. The absorbance
(OD) was measured at 650 nm by spectrophotometer. The results of the test were then expressed
as a ratio using the following formula: S/P% = ((ODsample – ODmean negative control)/(ODmean
positive control

– ODmean negative control)) x 100 . The test is considered valid if the difference

between the mean OD of the positive control and that of the negative control is greater than or
equal to 0.150. In addition, the OD of the negative control mean must be less than or equal to
0.20. Serum samples for N. caninum with S/P% of less than 50% are classified as negative and
the sample with S/P ratio greater or equal to 50% are classified as positive per manufacturer’s
instructions.
5.1.6 Tritrichomonas foetus hemolytic assay.
The test was performed as described previously.4 Briefly, 25µl of 1% fetal calf serum in
PBS was added to each well of a 96 well plate, then 25 µl of test serum at two fold dilution was
added to each well. Then 25µl 1% T. foetus antigen coated RBC addition to each well and the
plate was carefully shaken and incubated for 1hr. Later 50 µl reconstituted lyophilized guinea pig

54

complementf at a concentration of 1:10 was added to each well and was incubated for 1 hr at
room temperature and finally, the plate was shaken again, incubated for 90 min, and the results
were read visually. The extent of hemolysis was categorized subjectively as follows; 0-no
hemolysis; 1 - scant (25%); 2 - mild hemolysis (50%); 3 - moderate hemolysis (75%); 4 complete hemolysis (95-100%). Titers were reported as the highest serum dilution that gave at
least 75% hemolysis, i.e. a score of 3 or more.
5.1.7 Tritrichomonas foetus isolation
Vaginal and preputial secretions were collected from the vaginal fornix and the prepuce
using a dry, sterile Cassou artificial insemination pipette. The external surfaces of the vulva or
prepuce were cleaned of extraneous matter and the pipette aseptically inserted and used to scrape
the epithelial surfaces of the vagina or the prepuce to obtain sheets of epithelial cells and an
ample amount of secretion (approx. 3-6 ml.). The collected materials were inoculated into T.
foetus specific mediaa and incubated at 35oC in a 5% CO2. The cultures were examined daily for
7 days post-inoculation for any motile organisms and compared with positive controls incubated
simultaneously.
5.1.8 Statistical analysis
Association between the presence of antibodies to the different infectious diseases agents
were examined by the Chi-squared test on all pairs of the examined diseases. Correlations
between age, sex, and parity, and the ELISA and hemolytic assay results were examined by
Spearman’s correlation statistic.

55

5.2 Results
5.2.1 Herd information
The mean age of the females was 8.5 years (S.D. +/- 1.8), and that of the males was 4.2
years, (S.D. +/- 1.7). The mean calving interval of 2.5 yr was calculated based on the first
calving at 5 years and the number of calves produced as reported by the herdsman.
5.2.2 ELISA serology results
The IBR, BVDV, and BHV-4 ELISA results were positive in 69.0%, 13.0%, and 20.6% of
the total cattle (Table 5.1). Antibody against N. caninum was found in 6.1% of the total number
of the animals. Antibody against C. burnetii was confirmed in 17.7% of the animals, while 7.3%
were classified as suspect with an intermediate OD value. No antibodies against B. abortus were
detected in any of the animals tested (Table 5.2).
5.2.3 Hemolytic assay and culture results for T. foetus
The results of the hemolytic assay indicated that 47.6% of the animal had positive seroreactivity with 47.4% of the bulls being positive and 47.6% of the cows being positive; however,
no T. foetus organisms were isolated via culture from any animals in the herd (Table 5. 3).
5.2.4 Disease Associations
Potential associations between the investigated pathogens were examined and a significant
association was observed among the viral pathogens. A positive association was noted between
IBR and BVDV (chi-square with 1df = 6.810, p = 0.009), between IBR and BHV-4 (chi-square
with 1df = 6.028, p = 0.014), and between BVDV and BHV-4 (chi-square with 1df = 10.021, p =
0.002). No association was found between the bacteria and the protozoan agents with any viral
agent or each other.

56

Age of the cow was negatively correlated with BVD ELISA results (p = 0.0261). No bulls
were positive for BVD (different from cows, p < 0.001). There were no other correlations
between age, parity, or sex and any of the investigated pathogens. Only three cows were
pregnant at the time of testing; however, there was no consistent pattern of serological results
noted among them.
5.3 Discussion
This study examined a typical African herd and found evidence that these animals have
been exposed to, or were presently infected with, a wide range of important reproductive tract
infectious agents, and thus provides an instructive reminder that there is a need to focus on the
control of such infections in order to improve the contribution cattle make to the developing
economy of the rural sector.
This herd had an average calving interval of 2.5 years and as the time of our survey,
only 2.4% of the cows were pregnant, a common profile for cattle in this geographic area.
Herdsmen did not report abortions, but cows with reproductive infectious diseases often lose
their pregnancy early when it is difficult to observe, typically require more services per
pregnancy than their seronegative herd mates, and are more likely to be non-pregnant at any time
point.
This, the first serological analysis in Ghana, examines the prevalence of multiple agents
simultaneously, showed strong serological evidence that multiple important reproductive
infectious diseases are present in this herd; this is likely to be the case in many cattle herds in this
area. These infections are, in all likelihood, impacting livestock production in many parts of
Ghana and West Africa.

57

High seroprevalences of IBR, BHV-4 and T. foetus were detected with lower
seroprevalences of BVDV, Q fever, and N. caninum. Interestingly, there was no serological
evidence of brucellosis in this herd. This latter finding is contrary to the earlier findings from a
study conducted in a different region of Ghana carried out before significant nationwide efforts
had taken place to reduce the impact of this important disease.25 Literature reports on brucellosis
prevalence at individual and herd levels vary considerably across Africa: with estimates of
individual animal seroprevalence of 20.2% in Sudan, 18 between 0.3% and 8.2% in Eritrea, 24
12.3% to 14.1% in Tanzania,33 6.6% in Chad , 30 3.3% in Central African Republic, 21 14.1%
to 28.1% in Zambia , 20 and very recently, as low as 3.1% in Ethiopia. 17 At the herd level,
estimates range from 2.4% and 46.1% under different husbandry system in Eritrea.24 up to 80%
in Tanzania , 33 and in Zambia, from 46.2% to 74% . The failure to detect antibodies to B.
abortus in this herd could be due to improved management and control measures that has been
adopted since it was last measured in 1966.25
The analysis indicated almost half of the cows had a positive hemolytic titer to T. foetus;
though no T. foetus organism was cultured. This may be due to the low level of sensitivity of
culturing cows. 5. Furthermore, it is generally believed that most infected females clear the
organism within 2-5 months after infection. 7 Since calving intervals are so prolonged, the actual
time cows harbor the organism may be relatively short. It is well known that bulls are an
important component of any study addressing transmission and prevalence of reproductive
diseases in cattle. Age–related factors have been reported to be significant in the prevalence of
trichomoniasis in bulls, 5 and thus it is important to note that only a few bulls, and in fact, only
younger bulls were sampled in this study; these may not have been the main source of T. foetus
in this herd.

58

A significant association between the viral pathogens was detected, suggesting that similar
factors, such as animal density and compromised immunity, may be of importance in the spread
of this particular group of diseases. Further investigation of the interaction of these viruses may
be a fruitful area of investigation for improving Ghanaian and other regions livestock production.
This study focuses on a single herd in Ghana and is a first approach to provide the data on
the current status of reproductive infectious diseases necessary to improve the status of bovine
reproduction in this rural region of the world. It is necessary to extend these studies to greater
numbers of animals throughout Ghana and in other African countries. Infectious agents not
included in this study - such as leptospirosis, toxoplasmosis, and campylobacteriosis - very
likely could be present in African cattle herds and need also to be included in future studies, as
should other potential causes of poor fertility, such as nutrition. Comprehensive and nation-wide
survey and definitive isolation of these pathogens rather than serosurveillance are the logical
next steps to confirm the significance of these diseases. In addition to these microbiological
approaches, parallel studies to quantify the economic impacts of the pathogens on animal health
and production are crucial. The data from this study will be important in the design and pursuit
of additional studies and the rational development of local, national, and regional control
strategies for farmers, veterinarians, and policy-makers.
5.4 Acknowledgements
The authors thank Kwadwo Frimpong for his assistance in ELISA analysis and Mona
Abbas, Maxwell Quartey, and Ishmael Okine for their technical services. Drs. Scott Fitzgerald,
John Kaneene, and P. S. MohanKumar also provided valuable advice during the planning of this
project and Dr. Roger Maes and his staff assisted in the validation of viral ELISA assays.

59

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60

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Mcdermott, J. J. & Arimi, S. M. (2002) Brucellosis in sub-Saharan Africa: epidemiology,
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Moerman, A., Straver, P. J., De Jong, M. C., Quak, J., Baanvinger, T. & Oirschot, J. T.
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Nakoune, E., Debaere, O., Koumanda-Kotogne, F., Selekon, B., Samory, F. & Talarmin,
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Rodolakis, A., Berri, M., Hechard, C., Caudron, C., Souriau, A., Bodier, C. C.,
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Schelling, E., Diguimbaye, C., Daoud, S., Nicolet, J., Boerlin, P., Tanner, M. & Zinsstag,
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32.

Taylor, W.P., Okeke, A. N. & Shidali, N. N. (1977) Prevalence of bovine virus diarrhea
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33.

Weinhaupl, I., Schopf, K. C., Khaschabi, D., Kapaga, A. M. & Msami, H. M. (2000)
Investigations on the prevalence of bovine tuberculosis and brucellosis in dairy cattle in
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34.

Woldehiwet, Z. (2004) Q fever (coxiellosis): epidemiology and pathogenesis. Research
in Veterinary Science. 77, 93-100

35.

Yildiz, K., Kul, O., Babur, C., Kilic, S., Gazyagci, A. N., Celebi, B. & Gurcan, I. S.
(2009) Seroprevalence of Neospora caninum in dairy cattle ranches with high abortion
rate: special emphasis to serologic co-existence with Toxoplasma gondii, Brucella
abortus and Listeria monocytogenes. Veterinary Parasitology. 64, 306-310

63

Figure 5.1: Apolonia the study site, a village close to Accra, located in the coastal
region of Ghana

64

Table 5.1. ELISA serology results for infectious bovine rhinotracheitis (IBR), bovine virus
diarrhea (BVD), and bovine herpesvirus 4 (BHV-4) from 166 cattle from a herd in Apolonia, in
coastal Ghana.

Test Result

ELISA Seropositivity (% of Animals)
IBR

BVD

BHV-4

Male Female Total Male Female Total Male

Female Total

Negative

37.0

30.1

31.0

100

84.9

87.0

89.5 78.1

79.4

Positive

63.0

69.9

69.0

0.0

15.1

13.0

10.5 21.9

20.6

65

Table 5.2. ELISA serology results for Q fever (Coxiella burnetii), brucellosis (Brucella
abortus), and neosporosis (Neospora caninum) from 166 cattle from a herd in Apolonia, coastal
Ghana.
Test Result

ELISA Seropositivity (% of Animals)
C. burnetii

B. abortus

N. caninum

Male Female Total Male Female Total Male

Female Total

Negative

83.4

74.0

75.0

100

100

100

88.9 94.6

93.9

Positive

5.6

19.2

17.7

0.0

0.0

0.0

11.1 5.4

6.1

11.0

6.8

7.3

0.0

0.0

0.0

0.0

0.0

Suspect

a

a

0.0

“Suspect” refers to samples that were intermediate in reactivity and could not be accurately

interpreted.

66

Table 5.3. Hemolytic assay results for serum antibody against Tritrichomonas foetus in 166
cattle from a herd in Apolonia, coastal Ghana.

Test Result

Hemolytic Assay for Seropositivity to
Tritrichomonas foetus (% of Animals)
Male

Female

Total

Negative

52.6

52.4

52.4

Positive

47.4

47.6

47.6

67

Chapter 6

Cytokine responses to Tritrichomonas foetus infection in a mouse model
Abstract
Host immune factors are hypothesized to be important in determing the outcome
of Tritrichomonas foetus infection in cattle. Previous rodent model studies have shown
younger mice are more susceptible to T.foetus infection and that the success of infection
is determined in the first two days post-inoculation. This study was undertaken to
determine what factors underlie the different host responses that vary by age in this early
period. We hypothesize cytokines which are present in the early innate response vary
with the age of the host and are responsible for the variation in susceptibility to infection.
In this study, 4- and 8-wk-old BALB/cAnN mice inoculated intra-vaginally with 106
T.foetus organisms (D1 strain; T. foetus) were evaluated after 5 days of infection.
Susceptibility to infection; serum, vaginal, and uterine tissue cytokine profiles; and
histological appearance of the reproductive tract were determined and compared.
Successful T. foetus infection was determined by culture of vaginal and uterine flushes.
All 4-wk-old mice became infected following inoculation, but only 60% of the 8-wk-old
mice became infected. Vaginal levels of TNF were significantly higher in the inoculated
8-wk-old mice as compared to both inoculated and control 4-wk-old mice. In serum, by
contrast, TNF values were significantly higher in the infected 4-wk-old mice as compared
to control 4-wk-old mice, but similar to both infected and control older mice. IL-2 and
IL-4 levels in the 4wk infected were significantly lower when compared to their controls.
Histopathologically, there were no significant differences between the treatment groups

68

and controls. In summary, younger mice appear more susceptible to intravaginal infection
by T.foetus and the cytokine TNF may be involved in resistance to infection.
Introduction
Tritrichomoniasis is sexually transmitted disease of cattle caused by the protozoa,
Tritrichomonas foetus (T .foetus).16 Bovine trichomoniasis is an insidious, economically
damaging venereal disease of cattle with a worldwide impact especially in places where natural
breeding is practiced. 15 Trichomoniasis in the female host is characterized by mild endometritis
and occasionally by pyometra and abortion, while in the bulls, infections cause only mild
inflammation and usually ends in a perpetual carrier state. 19 The incidence of disease depends
to a large extent on management practices, such as using natural breeding and having older bulls
in the herd.
The economic effect of the disease is expressed in infertility; repeat breeding, delayed
return to oestrus after mating, early embryonic death and sometimes abortion. Increased
prevalence of T .foetus in a herd is associated with a significant reduction in calf crop revenue.
Currently, there is no treatment for trichomoniasis in cattle which is legal in the U.S., 15 and
infected animals are culled to control spread of the disease. 12
Studies aimed at understanding the host response in order to control and manage the
disease have focused on the acquired immune response. Results from these studies indicates
that, cows often recover from infection relatively quickly, which leads to partial but transient
immunity to reinfection. 25 In order to elucidate the mechanism of host immune response,
different animal models have been studied and the laboratory mouse has been considered a
useful model. 2, 7, 26 Very few of these studies have focused on the host immediate
and how it may impact the ability of T. foetus to establish an infection.

69

response

The innate immune response is key to understanding an antimicrobial challenge to a
mucosal organ. Generally, an innate immune response is initiated by binding of microbial
pathogen-associated molecular patterns (PAMPs) to Toll-like receptors ( TLR) located on
phagocytic and epithelial cells.3,27 As a result of PAMP-TLR binding, chemokines that attract
phagocytic cells to the affected site and secretion of type 1 interferons that attract and activate
natural killer (NK) cells occur. Epithelial cells in the female reproductive tract express TLR-4
and CD14 as well as TLR1, 2, 3, 5 and 6 which contribute protection.

10

CD4+ is a subset of T-cells known to contain both inflammatory T-cells and T-cells
responsible for helping antibody responses.1,6 The protection against T .foetus infection through
T-cell-dependent responses mechanism has not been well elucidated, although effective
immunity against most microbial pathogens, including many parasitic protozoans, is typically Tcell dependent. An example is effective immunity against Plasmodium sp. which requires CD4+
T-cell-regulated immune responses,

20,22,28

including T-cells that produce IFN-g and tumor

necrosis factor (TNF)-a.13 Effective immunity can sometimes be determined by which CD4+ Tcell pathway response predominates. A Th1 response against Leishmania spp., that involves
IFN-g and TNF-a, leads to a favorable outcome, where as a predominately Th2 response
exacerbates disease. 14,17,18,23 In contrast, Th2 responses characterized by IL-4, IL-5, and
production lead to more favorable outcomes with Giardia spp. infections.17, 23, 29
The Th1/Th2 pathway paradigm is not well defined in bovine protozoal infections as
immune and inflammatory responses are sometimes heterogeneous, inducing both Th1- and Th2like responses, whereas in other examples predominantly Th1 or Th2 responses occur. 6 For
example, T-helper responses induced in Th clones against the rhoptryassociated protein-1 of
Babesia bigemina are biased toward Th1, and are characterized by a strong IFN-g response and a

70

weak IL-4 response. 21 In contrast, clonal T helper responses in Fasciola hepatica-infected cattle
are characterized by strong IL-4 responses and weak IFN-g activity. 6
The study described here was undertaken to characterize the reproductive tract’s
immune response to protozoal infectious agents using a mouse model of trichomoniasis (Chapter
1; figure 1), addressing the hypothesis that the cytokine response to infection and success of
infection vary with age. The specific objective is to establish the cytokine environments during
early T.foetus infection in two different age groups of mouse and relate these to the success of
intravaginal infection.
6.1 Materials and methods
6.1.1 Mice
Forty BALB/c AnN female mice (National Cancer Research Institute), 4 and 8wks old,
were housed at the Vivarium of College of Veterinary Medicine in Michigan State University
according to the rules and regulations of IUCAC. Subjects were divided into groups of 10
composing of (1) 4-wk old control, (2) 4-wk old infected, (3) 8-wk old infected and, (4) 8-wk
old control. Animals were provided with controlled temperature quality. Light was provided on
12 hour light/dark cycle and food and water offered ad libitum.
6.1.2 Determination of T .foetus infection
Vaginal washes were obtained initially and at necropsy by aspiration with a micropipette
using 50 µl of sterile Phosphate Buffer Saline (PBS). To determine T.foetus infection, culture
aspirates were inoculated into Trypticase Yeast Maltose (TYM), incubated at 37o C and
examined daily microscopically daily for 7 days for the presence of T.foetus

71

6.1.3 Cytokine profile
Blood was collected from the saphenous vein, placing 0.3-0.5ml into 1.5ml cryovials
tube. The cryovials were centrifuged at 3000rpm for 5min, and serum was collected and stored at
-20 C until it was used.4
6.1.4 Estrous cycle determination
All animals were examined to determine the stages of estrous at the time of death.
Samples of vaginal mucus were obtained by washing with 50µl of sterile PBS and collecting the
sample by aspiration with a micropipette; samples were examined microscopically at 10x
magnification as previously described.5
6.1.5 Parasites
Tritrichomonas. foetus strain D1 was originally obtained from a cow with severe postcoital pyometra.24 This strain, at a intravaginal dose of 1 x 106 T. foetus organisms, was used in
several studies successfully intravaginally infecting 100% of experimental heifers .5,8 Parasites
were maintained in culture by using TYM medium that contained 10% fetal calf serum until
inoculation.9
A mouse-adapted strain of T. foetus (MuTF 52100V) was developed by first
intravaginally infecting BALB/ cAnNCr mice with strain D1. Then cultures from vaginal lavages
of these infected mice were pooled, grown in culture using TYM media, transferred to fresh
media every 2 to 3 days, and subsequently inoculated into susceptible mice. A second-generation
mouse-adapted strain (MuTF 10N) was developed by selecting positive cultures from mice
infected with strain MuTF 52100V. For inoculation, this material was centrifuged at 3000rpm for
10 min and the sediment/pellet washed 2X with PBS. These were then resuspended into PBS and
their concentration was standardized.

72

6.1.6 Experimental Design
After ear-notching and initial blood sampling, female mice were allowed to acclimate for
2 days before the experiment began. Females in each group were intravaginally inoculated with
10 4 motile T. foetus strain MuTF10N using organisms suspended in 10 ml trypticase-yeast
extraction serum (TYI) media by using an Eppendorf pipette; control animals were sham
inoculated with sterile TYI media. 2 A second inoculation of T. foetus or TYI at the same dose
and volume was given the following day. Animals were killed 5 days after inoculation. At
necropsy, they were weighed and blood was collected by cardiac puncture. Before removal of
the reproductive tract, the vagina was flushed with 80 µl of sterile PBS from which 20 µl was
cultured in TYM and the remainder was frozen at -80 oC. After removal of the whole
reproductive tract, the uterus was separated from the vagina at the cervico-vaginal border, and
was bisected. One uterine horn was flushed with 80 µl of sterile PBS and cultured in TYM. This
uterine horn and its ovary were frozen at -80 oC and the contralateral horn and ovary were fixed
in 4% buffered paraformaldehyde. Paraformaldehyde fixed tissues were transferred to PBS after
24hrs, then processed through gradients of progressively more concentrated ethanol, until the
tissues were stored long term in 70% ethanol. Cultures were examined microscopically and
successful infection was determined by the presence of motile parasites.
6.1.7 Histopathology and Immunohistochemistry
Paraformaldehyde-fixed tissues were trimmed, embedded in paraffin, sectioned at 5-7 μm
and routinely processed for immunohistochemistry. An enhanced V Red (Alkaline Phosphatase
Red) Detection System, (Ventana Medical Systems, Inc., Tucson, AZ, USA) as well as bulk
buffers specifically designed for use on the BenchMark Automated Staining System, (Ventana
Medical Systems, Inc., Tucson, AZ, USA) were used for immuno-labeling and visualization.

73

Slides were baked in a drying oven at 60°C for 30 minutes. The slides were then barcode labeled,
and placed in the BenchMark for deparaffinization and heat-induced epitope retrieval. Antigen
retrieval was performed using retrieval solution CC1 (Medical Systems, Inc., Tucson, AZ, USA)
with an 8-minute heating and an 8-minute cooling cycle. A rabbit -polyclonal anti-T. foetus
antibody at a concentration of 1:500 for 32 minutes was used as a primary antibody. A
secondary antibody, goat anti-rabbit IgG, was selected to avoid cross reactivity between
irrelevant mouse antibodies. The slides were counterstained using Ventana hematoxylin
(Ventana Medical Systems, Inc., Tucson, AZ, USA), and bluing for 2 minutes each, then
dehydrated, cleared and mounted. Formalin-fixed, paraffin-embedded sections of uterus from a
T. foetus infected cow that had previously been tested for trichomoniasis were used as positive
controls. Use of an irrelevant primary antibody was used as a negative control.
6.1.8 Flow cytometry
Vaginal and uterine washes were assayed for IL-2, IL-4, IL-5, IFN-γ and, TNF-α
simultaneously using the mouse Th1/ Th2 – cytokine cytometric bead array (CBA) kit (BD
Biosciences). Briefly 50 µl of each sample was mixed with 50 µl of mixed capture beads and 50
µl of the mouse Th1/Th2 PE detection reagent made up of PE-conjugated anti-mouse IL-2, IL-4,
IL-5, IFN-γ and, THF- α . The samples were incubated at room temperature for 2 hours in the
dark. Following incubation, 1 ml of buffer was added to samples and centrifuged at 200x g for 5
minutes. The remaining wash buffer was aspirated and discarded from each assay tube and the
pellet resuspended in 300 µl of wash buffer. Samples were run using BD FACS Calibur Flow
Cytometer and data analyzed using the CBA Analysis software (BD Bioscience).

74

6.1.9 Preparation of Vaginal and Uterine homogenates
Vaginal and uterine homogenates were analyzed using the same procedure as described
above, except that tissues were homogenized and their protein content quantified first, using a
spectrophotometric assay. Briefly, frozen tissues were ground to a fine powder by hand in a
mortar 1/3 filled with liquid nitrogen; this was then resuspended in a homogenate buffer (PBS +
0.05% NP-40 non-ionic detergent + anti-protease tablet). The protein quantity was measured
using with a spectrophotometer at a wavelength of 750λ. After flow cytometric assay, cytokine
values were adjusted for the respective protein content.
6.1.10 Statistical analysis
The frequencies of successful intravaginal infections in 4 wk and 8 wk old infected mice
were expressed as percentages. Statistical analysis of differences in the means between cytokine
levels in infected young and adult mice, and between infected and uninfected age –matched
controls was performed using the Mann Whitney test (one tailed) based on the hypothesis that
cytokine levels would increase in response to infection. In each experiment, a p value ≤ 0.05
between infected young and adult mouse was considered significant.
6.2 Results
6.2.1 Stage of estrus
The stages of estrus at the time the mice were killed ranged from proestrus to metestrus.
Most of the subjects from both treatment and control groups of both ages were in proestrus,
followed by estrus, and then metestrus. No animal was found in diestrus (Table 6.1).
6.2.2 Percentage of vaginal and uterine Infection of T. foetus in young and adult mice

75

The overall frequency of infection in the younger mice was 100% with vaginal infections
being more common than uterine; while the frequency of infection in the older mice was 60%,
with the uterus more commonly infected (Figure 6.1)
6.2.3 Analysis of cytokines levels
An initial comparison of the levels of all measured cytokines, from all infected mice
regardless of age, with the control group regardless of age, showed that there was no significant
difference between the two groups (data not shown). However, significant differences in some
cytokine levels were noted between the different age groups.
The TNF-alpha concentration from vaginal homogenates was significantly higher in the
8 -wk old infected mice as compared to the 4 wk old infected mice (p= 0.032) (Table 6.2;
Figure 6.2). IL-2 and IL-4 concentrations from vaginal homogenates were significantly higher
in the 4 wk old control mice as compared to the 4 wk old infected (p= 0.0477 ) and IL-4 ( P=
0.0412 ), respectively (Table6. 2; Figure6. 3 and 6.4) . The uterine homogenates were also
significantly higher for IL-2 and IL-4 in the 4wk old control as compared to the 4 wk old
infected mice with p values of 0.0428 and 0.0063, respectively (Table 6. 3; Figure 6. 5 and 6.6).
The TNF-alpha concentration in the serum sample was significantly higher in the 4 wk old
treated mice compared to the 4 wk old control group ( p=0.0158 ) (Table 6.4 ; Figure 6.7);
however, there was no significant difference between the 8 wk old infected and its respective
control.
6.2.4 Histopathology and Immunohistochemistry
Positive labeling of organisms with the specific anti-T.foetus antibodies was seen in the
lumen of the mouse’s vagina and occasionally mixed with the mucinous secretion (Figure 6. 8).

76

No positive labeling was seen deeper in the tissues. Histopathologically, there was no significant
lesion other than presence of some T.foetus organisms in the vaginal lumen.
6.3 Discussion
The results of this study confirm that younger mice are more susceptible to T. foetus
infection than older mice and the cytokine environment within the reproductive tract in acute
T.foetus infection also varies with the age of the host.
In our studies, the 4 wk old mice had a higher frequency of infection, although
following successful infection extension to the uterus was more common in the 8 wk old mice.
However, in all groups, cellular inflammation and histopathology was generally absent at this
early stage of infection. This study also shows that vaginal TNF-α is elevated more in the 8 wk
old mice compared to uninfected 8 wk mice and all the younger mice (both infected and
uninfected). We also showed that there was a decrease or down regulation of IL-2 and IL-4
respectively in the 4wk old infected mice compared to the uninfected mice and all the adult mice
(both infected and uninfected).
Interestingly, the serum levels of TNF- α are initially low in the uninfected 4 wk mice,
but increase in response to infection in these animals to levels similar to those seen in both
infected and control 8 wk mice. This suggests that serum TNF- α levels may be constitutively
higher in older mice providing some protection during T. foetus infection. Similarly, the data
suggests that TNF- α in vaginal homogenates tends to increase with infection in younger
animals and is higher in older animals both uninfected and significantly in infected mice.
Our study demonstrates a phenotype in mice in which TNF- α is higher and IL-2 and IL-4
resists degradation or is downregulated in older animals inducing an increase in the resistance to
infection. TNF-α has been confirmed to play a protective role in host resistance against

77

microbes. It is possible that T. foetus infection of a mucosal surface activates Toll-like Receptors
(TLRs) which in turn activate transcription factors (including NF-κB, Egr-1 and AP-1), which
bind to the TNF promoter.4 Following activation of the promoter and subsequent production,
TNF-α protects by activating neutrophils via IL-8. TNF-α also activates macrophages, and
generates both a respiratory burst and nitric oxide (NO) dependent killing pathways. TNF-α may
also induce natural killer (NK) cells to produce interferon-gamma, a significant early host
defense mechanism.4, 11
These findings suggest that TNF- α and Th2 cytokines are important in early resistance.
Additional studies are needed to identify the specific pathways and effector molecules that these
cytokines govern and to apply these findings to preventative and therapeutic strategies.
6.4 Acknowledgements
This work was supported by the Cowham grant of the Department of Pathobiology and
Diagnostic Investigation, College of Veterinary Medicine, MSU. The authors thank Lori
Bramble for her assistance in cytokine analysis and Jennifer Lamoureux for her technical
services.

78

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82

Table 6. 1 Stages of estrus cycle of mice on the 5th day of post infection
with T.foetus as determined by vaginal cytology and histopathology post-mortem

Number of mice in each group
4week old
Stages of

8week old

Treated

Control

Treated

Control

Proestrus

6

5

4

7

Estrus

2

3

4

2

Metestrus

2

2

2

0

Diestrus

0

0

0

0

estrous cycle

83

100

8wk mice(% vaginal infected )
8wk mice(% uterine infected)
4wk mice (% vaginal infected)
4wk mice(% uterine infected)

Percentage

80
60
40
20
0

Figure 6.1. The percentage of T. foetus infected mice that were successfully infected in either
the uterus or the vagina. Note that all 4-wk-old mice had vaginal infections and only three
uterine after inoculation. In comparison only 60% (6) of the 8-wk-old mice had vaginal
infections and four uterine.

84

Table 6.2. Vaginal homogenate cytokine concentration in T.foetus-infected and control mice (4
wk and 8wk old) 5 days post-inoculation (mean and standard error of the mean (SEM) values)
IL-2

8 wk cont

TNF-α

0.4300

2.550

1.730

1.020

5.030

(0.3839)

(0.4169)

(0.2555)

(2.495)

1.263

3.500

2.413

1.438

3.288

(0.4285)

(0.6043)

(0.2456)

(0.8794(

0.9667

2.700

2.600

1.544

8.356

(0.3308)

8 wk inf

IFN-γ

(0.3909)

4 wk cont

IL-5

(0.3077)

4 wk inf.

IL-4

(0.2687)

(0.5819)

(0.1260)

(2.074)

0.8286

2.686

1.657

1.314

5.314

(0.4004)

(0.2198)

(0.5200)

(0.1100)

(1.392)

85

TNF15

b
10

pg/ml

a

a,b
a,b

5

co
nt

in
f
8

w

k

w
k
8

k
w
4

4

w

k

in
f

co
nt

0

a, b-Different superscript indicate significantly different TNF-α levels in tissue
Figure 6. 2. The levels of the cytokine TNF-α in vaginal homogenates at 5 days post-infection
with T. foetus. A significant difference is seen between the 8 wk old infected and the 4 wk old
infected. Bar represents SEM. (n=10 in each group), p=0.032.

86

IL-4
5

b

pg/ml

4

a,b

a

3

a,b

2
1

co
nt

in
f
k
8

w

k

w
8

k
w
4

4

w

k

co
nt

in
f

0

a, b-Different superscript indicate significantly different IL-4 levels in tissue
Figure 6.3. The levels of the cytokine IL-4 in vaginal homogenates at 5 days post-infection
with T. foetus. A significant difference is seen between the 4 wk old infected and the 4 wk old
sham control (p=0.0477). Bar represents SEM. (n=10 in each group).

87

IL-2
2.0

b
pg/ml

1.5

a,b

a,b

1.0

a
0.5

co
nt

in
f
k
8

w

k

w
8

k
w
4

4

w

k

co
nt

in
f

0.0

a, b-Different superscript indicate significantly different IL-2 levels in tissue
Figure 6.4. The levels of the cytokine IL-2 in vaginal homogenates at 5 days post-infection with
T. foetus. A significant difference is seen between the 4 wk old infected and the 4 wk old
sham control (p=0.412). Bar represents SEM. (n=10 in each group).

88

Table 6.3. Uterine homogenate cytokine concentrations in T.foetus-infected and control mice (4
wk and 8wk old) 5 days post-inoculation (mean and standard error of the mean (SEM) values)
IL-2

8 wk cont

TNF-α

0.2900

1.760

0.8900

0.7800

0.7400

(0.2310)

(0.3096)

(0.2154)

(0.3103)

1.013

2.700

1.750

1.175

1.188

(0.2809)

(0.5751)

(0.3239)

(0.3796)

0.6500

2.240

1.250

1.130

1.750

(0.2684)

8 wk inf

IFN-γ

(0.3232)

4 wk cont

IL-5

(0.1935)

4 wk inf.

IL-4

(0.3925)

(0.3478)

(0.2155)

(1.834)

0.7429

2.171

1.500

1.257

2.571

(0.2644)

(0.1700)

(0.3970)

(0.06494)

(1.222)

89

IL-4
4

b

pg/ml

3

a,b

a,b

a

2
1

co
nt

in
f
k
8

w

k

w
8

k
w
4

4

w

k

co
nt

in
f

0

a, b-Different superscript indicate significantly different IL-4 levels in tissue
Figure 6.5. The levels of the cytokine IL-4 in uterine homogenates at 5 days post-infection with
T. foetus. A significant difference is seen between the 4 wk old infected and the 4 wk old
sham control (p=0.0428). Bar represents SEM. (n=10 in each group).

90

IL-2
1.5

b
a,b

a,b

pg/ml

1.0

a

0.5

co
nt

in
f
k
8

w

k

w
8

k
w
4

4

w

k

co
nt

in
f

0.0

a, b-Different superscript indicate significantly different IL-2 levels in tissue
Figure 6.6. The levels of the cytokine IL-2 in uterine homogenates at 5 days post-infection with
T. foetus. A significant difference is seen between the 4 wk old infected and the 4 wk old sham
control (p= 0.00063). Bar represents SEM. (n=10 in each group).

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Table 6.4. Serum cytokine concentrations in T.foetus-infected and control mice (4 wk and 8wk
old) at 5 days post-inoculation (mean and standard error of the mean - SEM)
IL-2

8 wk cont

TNF-α

1.190

1.380

3.530

1.060

6.500

(0.4809)

(0.3467)

(0.2459)

(0.5160)

0.8857

0.5857

2.614

0.4143

4.129

(0.3795)

(0.5059)

(-0.2450)

(0.7981)

1.250

1.280

3.120

1.130

6.060

(0.1586)

8 wk inf

IFN-γ

(0.3240)

4 wk cont

IL-5

(0.2105)

4 wk inf.

IL-4

(0.3620)

(0.2210)

(0.2044)

(0.5476)

1.563

2.013

3.475

1.163

6.100

(0.1463)

(0.5191)

(0.3793)

(0.2890)

(0.3571)

92

TNF8

a

a,b

a,b

6

pg/ml

b
4
2

co
nt

in
f
k
8

w

k

w
8

k
w
4

4

w

k

co
nt

in
f

0

a, b-Different superscript indicate significantly different TNF-α levels in tissue
Figure. 6.7 Levels of the cytokine TNF-α in serum 5 days after infection with T. foetus. There
was significant difference between the 4 wk old infected and the 4 wk old sham control
(p=0.0158). Bar represents SEM. (n=10 in each group)

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Figure 6.8. Tritrichomonas organisms in the lumen of the mouse’s vagina, and occasionally
mixed with the mucinous secretion, as detected by immunlabelling with anti-T.foetus antibody.
Alkaline phosphatase red with hematoxyln counterstain

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Chapter 7

Potentially protective dual oxidase enzymes (DUOX1 and DUOX2) in the bovine reproductive
tract
Abstract
Vaginal and uterine mucosa is the portal for sexually transmitted viral, bacterial, and
parasitic diseases (STD) in animals and man. The sequelae of STD lead to major economic
costs in animal production and human medical care. Understanding the innate immunity
response at the mucosal surface of infection is essential to enhancing protection and
controlling STD. The recent discovery of the dual oxidases (DUOX1 /DUOX2) in different
mucosal membranes, and recognizing the phenomenon of H2O2 associated killing of
microbes, suggests that these enzymes may be important in the innate defenses of the
reproductive tract. DUOX1 and 2 have been recently identified in the reproductive tract of
mice; however, their role in the bovine reproductive tract has not been documented. The
aim of the current study is to: 1) investigate the presence of DUOX1 and 2 in the
reproductive tract of cows using immunohistochemistry (IHC) and rtPCR, and 2) examine
the production of DUOX1 and 2 in vitro during acute infection of cultured endometrial cells
with T.foetus by IHC and PCR, testing the hypotheses that the DUOX molecules exist in the
cow and increase in response to infection. Ovaries, oviduct, uterine mucosa, vagina, and
intestine were collected in RNAlater from 6 female cattle for analysis of DUOX1 and 2 gene
expression normalized to the endogenous control 18S gene. For IHC, samples were labeled
with DUOX1 and 2 monoclonal antibodies and examined for specific binding. A
comparison of mean critical threshold values (delta Ct) showed the highest DUOX1 and 2

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gene expression in the vagina and the lowest in the ovary. DUOX1and 2 expression was also
detected in both bovine trophoblast and endometrial cell lines. Bovine endometrial cells
were infected with T.foetus for 6 hours, however there was no significant change in DUOX1
and 2 levels, suggesting that if these molecules play a role in innate immunity, it may be
later than 6 hrs post infection.
Introduction
The vaginal and uterine mucosa is the portal of entry for sexually transmitted diseases
(STD), such as trichomoniasis in cattle caused by Tritrichomonas foetus. Tritrichomonas . foetus
is an extracellular organism and the infection in cows is generally self-limiting, though the
economically damaging effects of early embryonic death, infertility, and pyometra can be
devastating. Historically, research has focused on the role of acquired humoral immune
responses. However, few studies investigated how the innate reproductive immune response
contributes to protection against T. foetus and how these responses might influence initial
colonization. One study evaluated the effects of nitric oxide, a component of the early innate
immune response, in which it was shown to be an effector of macrophage-mediated cytotoxicity
towards the related human pathogen, Trichomonas vaginalis.26 This study was the first to
demonstrate that reactive nitrogen species (RNS) were able to kill the extracellular venereal
parasites; though, this did not implicate RNS as being a critical molecule in the establishment of
trichomonad infection within the reproductive tract.
Mechanisms that initiate immune responses and inflammation during T. foetus infection
have been rarely studied. In studies of T. vaginalis stimulation of Toll-like receptor 4 (TLR-4)
has been demonstrated, pointing to a mechanism whereby early inflammation is induced.34
Related studies with T. vaginalis have demonstrated that, the parasite has the ability to induce

96

IL-8 production from both human monocytes and human neutrophils. 27, 29 Similar processes can
be postulated to occur in the early response to T. foetus.
Recent studies have shown that dual oxidase molecules (specifically DUOX1 and
DUOX2), members of the NADPH oxidase family, which are expressed on the mucosal surfaces
of multiple mucosal membranes provide a well-defined, controllable, and dependable source of
reactive oxygen species (ROS) (e.g., HOCl, peroxinitrate, hydrogen peroxide), which function in
the early innate host response by catalyzing lactoperoxidase to generate antimicrobial
hypothiocynate. Reactive oxygen species produced by these DUOX enzymes and their byproducts can directly oxidize biomolecules in invading microbes in a nonspecific manner,
resulting ultimately in molecular damage and microbial cell death. 17
Studies have also demonstrated that DUOX1 is associated with specific cytokine
pathways. In fact, DUOX1 has been associated primarily with a Th2 cytokine response
characterized by IL-4, IL-5, and IL-8; whereas DUOX2 has been more closely associated with a
Th1 response, characterized by increases in IFN- γ DUOX1 mediates the production of
epithelial mucins, like MUC1 and MUC5AC, in response to inflammatory mediators such as
neutrophil elastase or tumor necrosis factor (TNF)-α16,30 In other studies, DUOX1 was found
as a mediator in epithelial production of the neutrophil chemokine IL-8 in response to
stimulation by bacterial lipopolysacharide (LPS) .25 Induction of DUOX enzymes has also been
described. DUOX1 production is induced in response to

IL-4 and IL-13 in respiratory tract

epithelium. DUOX2 expression is induced in response to IFN- γ in respiratory tract epithelium,13
as well as in response to insulin in thyroid cell lines .22 In another study, when DUOX enzyme
was silenced, it led to increased mortality and infection by gut microbes. This effect was

97

reversed by reintroduction of the DUOX enzyme, causing infection levels and mortality to fall,
demonstrating the key role of DUOX in mucosal immunity.12
The role of DUOX1 and DUOX2 in host immune responses has been well studied in the
respiratory and gastrointestinal mucosa. However, there is paucity of such information
regarding the situation in the reproductive tract mucosa. Recently, the presence of DUOX1 and
DUOX2 has been demonstrated in the mouse reproductive tract, including the vagina, uterus,
oviduct, and ovary (Agnew et al, manuscript in preparation). However, these molecules have not
been demonstrated in a livestock species, nor has the response of these molecules to a venereal
pathogen been investigated.
Tritrichomonas foetus, the causative agent of bovine trichomoniasis, is an extracellular
organism that typically produces Th2 response. Because, it has been demonstrated that Th2
cytokines regulate DUOX1 expression, it is expected that during T. foetus infection, DUOX1 and
Th2 cytokines production will be important for host protection. We hypothesize that DUOX1
and DUOX2 are present in the bovine reproductive tract and that DUOX1plays a key role in
early resistance to venereal infection by T. foetus.
The aim of the current study is to investigate the presence and production of DUOX1 and
DUOX2 in the bovine reproductive tract and in bovine endometrial cell cultures (Chapter 1;
figure1). This study also hypothesizes that in the presence of T. foetus DUOX1 increases in
bovine endometrial cell cultures using immunohistochemistry (IHC) and RT-PCR. The specific
objectives are;
1. To demonstrate the presence of DUOX1 and DUOX2 expression in the bovine
reproductive tract (in vivo).

98

2. To confirm the presence of DUOX1 AND DUOX2 expression in bovine endometrial
and trophoblast cell lines (in vitro)
3. To show an increase in DUOX1 expression in T. foetus-infected bovine endometrial
cells (in vitro).
7.1 Materials and methods
Subject material
7.1.2 Cow reproductive tract samples
Reproductive tract tissues (ovary, oviduct, uterus, and ovary) and ileum from six adult
cows were collected immediately after slaughter for immunohistochemistry (IHC) and realtime polymerase chain reaction (RT-PCR) investigation. For RT-PCR, approximately 2-4gm
each of ovary, oviduct, uterine mucosa, vagina, and intestine were collected and stored
separately in a centrifuge tube filled with 2mls of RNAlater (Qiagen, Valencia, CA) and
processed for RNA extraction. For IHC, 4mm thick portions of the above listed tissues were
collected into 15mls of 4% Paraformaldehyde.
7.1.2 Endometrial and trophoblast cell lines
The endometrial and trophoblast cell lines were kind donation from Dr. Linda Munson at
the University of California Davis.23,24 Cultures younger than 15 passages were seeded at 5x104
cells/ml/5cm2 density and maintained in DMEM-F12 (Gibco, Life Technologies, 3175 Staley
Road NY) medium, 5 µg/ml bovine iron-saturated transferring (ITS; Sigma-Aldrich), 10 ng/ml
murine epidermal growth factor (Becton-Dickenson, San Jose, CA), and 10% fetal bovine serum
(Invitrogen) at 37°C in 5% CO2. Upon 80-90% confluency the cells were harvested with 0.25%
trypsin with gentle rocking to dislodge the cells. For long term storage for later analysis cells

99

were suspended at the range from 2.9 x10 7 to 5.2 x 10 7 cells/ml in 1:1000 25-mercaptanol RLT
buffer (QIAGEN, Valencia, CA 91355) and then stored at -80 °C.
7.1.3 Infection of endometrial cell lines with T. foetus
The T. foetus isolate was originally obtained from a cow with severe post-coital
pyometra, and was kind donation from Dr. R. BonDurant (UC Davis) and maintained in culture
using trypticase-yeast extract (TYM) medium containing 10% fetal serum. 8 Endometrial cells
and trophoblast were grown to 75-90% confluence in 75 cm2 plates. One milliliter of T. foetus
innoculum was added to the media at the concentration of 1 x 106 cells/ml (multiplicity of
infection = 0.1 (MOI)), and the cultures were incubated as previously described for an additional
6 hours. The cells were trypsinized, counted, mixed with 25-mercaptanol/ RLT buffer, and
frozen as described above. Each plate infection was run in duplicate, and each experiment was
repeated three times.
7.1.4 RNA preparation
RNA was extracted using RNEasy Mini Kits and QIAshredder columns according to the
manufacturer’s instructions (Qiagen, Valencia, CA). The concentration of the obtained total
RNA was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific,
Wilmington, DE). RNA purity was determined by the A260/280 value, with ideal numbers
ranging between 1.8 and 2.1. The A260/230 ratio (which detects ethanol and salt contaminants
from RNA isolation) was provided as an additional measure of sample purity. A value of 1.0 or
greater is acceptable, with optimal values being1.8 or greater. All samples were between 1.97
and 2.09 for the A260/280 value, and all A260/230 values were greater than 1.8 except 3
specimens which had values of 0.35, 1.36, and 1.77.

100

7.1.5 Primer and probe development
Since Applied Biosystems does not offer off-the-shelf gene expression assays for
bovine DUOX1 or bovine DUOX2, custom assays were designed from predicted Bos taurus
mRNA sequences available in GenBank (XM 587550 and XM 001253634, respectively).
DUOX1 and DUOX2 cDNA sequences were analyzed with the NCBI BLAST program to
identify regions of the sequence that are similar between the 2 target genes. The gene
expression assays were designed in regions that did not show any similarities. Two assays
(primers & probe) were designed for each gene by Applied Biosystems and are proprietary.
PCR was run for DUOX1 and DUOX2 using a wide range of cDNA templates (0.32ng to
80ng) synthesized from 1ug of RNA. cDNA samples from ten tissues (Intestine 6 and 7; Ovary
10 and 11; Oviduct 7 and 10; Uterine Mucosa 10 and 12; Vagina 7 and 10) were synthesized for
serial dilution plates. This was carried out in order to determine the optimal amount of cDNA
template for efficient PCR amplification. Optimal PCR amplification was achieved with 20ng of
cDNA template.
7.1.6 cDNA Synthesis

cDNA was synthesized by RT- PCR of 2µg each total RNA using High Capacity cDNA
Reverse Transcription Kit according to manufacturer’s instructions (Applied Biosystems) and
primers. To eliminate amplification of any contaminating chromosomal DNA, the isolated RNA
was treated with RNase free DNase (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. Each cDNA sample was then amplified with Taq polymerase.
7.1.7 q PCR Processing
PCR data quality was assessed using a combination of factors, including visual analysis
of the shape of the raw PCR curve and the Ct value. Ct values less than 30 indicated high quality

101

data and correlate with samples that have high expression levels; Ct values from 30-35
corresponded to lower expression levels and Ct values over 35 were consistent with very low
expression levels. Ct values were normalized to the endogenous control (Ct target – Ct
endogenous control) to generate a delta Ct (DCT) value for each sample. DCT values were then
compared among the different tissue groups to identify statistically significant gene expression
differences. The endogenous control was 18S, based on extensive analysis for the most
consistent endogenous control from the various tissues (data not shown).
7.1.8 Immunohistochemistry for DUOX1 and DUOX2
Sections of ovaries, oviduct, uterine mucosa, vagina, and intestine were initially fixed in
4% paraformaldehyde, then after 24 hrs, rinsed in PBS. After another 24 hrs, the tissues were
placed in 30% ethanol, then 50% ethanol 1 day later, and then finally stored in 70% ethanol 1
day later. Samples stored in 70% ethanol were embedded in paraffin, and sectioned on a rotary
microtome at 4µ. Sections were placed on slides coated with 3-aminopropyltriethoxysilane and
dried at 56 C overnight. The slides were subsequently deparaffinized in xylene and hydrated
through descending grades of ethyl alcohol to distilled water. Slides were placed in Tris buffered
saline (TBS) pH 7.5 for 5 minutes for pH adjustment. Endogenous peroxidases were blocked
utilizing 3% hydrogen peroxide/ methanol bath for 20 minutes followed by a distilled water
rinse. Slides were then placed in TBS + Tween 20 (Thermo Fisher Scientific, Rockford, IL
61105) and stained with standard Avidin –biotin complex. Staining steps were performed at
room temperature on DAKO Autostainer. After blocking non-specific protein with normal Goat
serum and bovine serum (Vector Labs-Burlingame, CA) for DUOX1 and DUOX2 respectively,
for 30 minutes, sections were incubated with Avidin /Biotin blocking system for 15 minutes
(Vector Labs-Burlingame, CA). Following subsequent rinsing in TBS + Tween 20, for DUOX1,

102

slides were incubated for 30min with Polyclonal rabbit antibody DUOX2 ( Santa Cruz-Santa
Cruz , CA), diluted 1:200 with Normal Antibody Diluent (NAD) (Scyteck-Logan, UT). Slides
were then rinsed in two changes of TBS +Tween 20. After rinsing slides were incubated in
Biotinylated Goat and anti Rabbit 1gG H+ L (Vector) in NAD diluted 11ug/ml for 30min. In the
case of DUOX2, slides were incubated for 30 min polyclonal goat antibody DUOX2 (Santa
Cruz-Santa Cruz, CA), diluted 1:25 with Normal Antibody Diluent (NAD) (Scyteck-Logan, UT).
Slides were then rinsed in two changes of TBS +Tween 20. After rinsing, slides were incubated
in biotinylated bovine anti-goat IgGH +L (Vector) in NAD diluted 11ug/ml for 30 minutes.
After this, slides were rinsed in TBS + Tween 20 followed by the application of R.T.U.
Vectastain Elite ABC Reagent (Vector) for 30min. The slides were rinsed with TBS + Tween 20
and developed using Nova Red (Vector) for 15 min. At the completion of Nova Red, slides were
rinsed in distilled water, counterstained using Grill 2 Hematoxylin (Thermo Electron –
Pittsburgh, PA) for 30 sec, differentiated in 1% aqueous glacial acetic acid, rinsed in running tap
water. Slides were then dehydrated through ascending grades of ethyl alcohol; cleared through
several changes of xylene and cover slipped using Flotex permanent mounting media (Thermo
Scientific –Pittsburgh, PA).
7.1.9 Data Analysis
Data analysis was carried out using Applied Biosystems RQ Manager software v2.1)
and StatMiner software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
California 94404, USA). Data analysis was carried out by firstly comparing the mean DCT
values for each of the tissue types (for each gene). Ranked and arranged from highest to
lowest expression level, and secondly gene expression was measured using the relative
quantitation (RQ) method according to Applied Biosystem’s recommendations. In this

103

study, the gene expression level of each tissue was compared to the expression level present
in the intestine (control tissue).
7.1.10 Statistical analysis
Three experiments were conducted and each experiment was duplicated.
Paired and unpaired t-tests were used to compare control samples with test samples (cultured
with T. foetus). To determine changes in RNA concentration between each experiment, we
compared each duplicate test (cultured with T. foetus) with their respective controls for any
significant difference. To determine whether total amount of RNA changed was significantly
different between the T. foetus infected cells and the non- infected cell, each experiment in
duplicate, was reported as an average of the three, and unpaired t-test was used to compare any
significant difference.
7.2 Results
7.2.1 PCR Analysis: Ct Values Generated for Each Gene and Tissue
All three genes (DUOX1, DUOX2 and 18S) were present in each of the samples. The
18S was shown to be highly expressed, indicated by a ct value less than 30. DUOX1 expression
was more abundant than that of DUOX2 in the bovine reproductive tract, especially in the vagina
(Figures 7.1, 7. 2, 7.3).
7.2.2 Cellular Localization of DUOX Protein in Bovine reproductive tract
Positive labeling by the anti-DUOX1 and DUOX2 antibodies was seen in the cytoplasm
of the reproductive epithelium (Figure 7.4 A, B, C, D). The majority of the staining was
observed both in the apical plasma membrane and to a greater extent, in the apical portion of
epithelial cells.

104

7.2.3 In vitro cell results
Preliminary studies showed the presence of DUOX1 and DUOX2 by either qPCR or IHC
in the endometrial and trophoblast cells. ( figure 7.5A, B and C). Neither DUOX1 nor DUOX2
was found in the pure culture of T.foetus or in the cell culture medium.
7.2.4 Cellular Localization of DUOX Protein in Bovine endometrial and trophoblast cell lines
Moderate positive labeling by the anti-DUOX antibodies was seen within the cytoplasm
of the endometrial cells and weak staining in the trophoblast cells. ( figures 7. 6A and B).
7.2.5 Tritrichomonas foetus infected bovine cell lines
All three genes (DUOX1, DUOX2 and 18S) were present in each of the cell culture
samples. The 18S was shown to be highly expressed indicated by lower ct value less than 30.
DUOX1 and DUOX2 were shown to be expressed; however; there was no statistically
significant difference in the expression of DUOX1 or DUOX2. (Figures 7.7 and7. 8)
7.3 Discussion
Our investigation has demonstrated for the first time, using PCR and
immunohistochemistry, the presence of DUOX molecules in the bovine reproductive tract, and in
bovine trophoblast and endometrial cell lines. Based on our assays, DUOX1 was found to be
more abundant than DOUX2 in the reproductive tract. We were not able to show, however, a
significant change in either DUOX1 or DUOX2 expression after cells were treated with live T.
foetus organisms after 6 hr of co-incubation. This suggests that, if DUOX molecules play a role
in the immediate immune response to T. foetus, this occurs sooner or later than 6 hrs after
inoculation. The 6 hrs time course was selected based on the results of our preliminary studies,
in which 6hrs T. foetus infected endometrial cells showed significant DUOX1 expression
comparing to that of 12hrs and 24 hrs. Also, this is supported by the previous studies in

105

demonstrating the host defense role of DUOX in Drosophila by Ha EM et al (2005) in which The
host defense role of DUOX was demonstrated in Drosophila when the DUOX homologue,
dDUOX, was silenced followed by microbes infection in the gut. After, 6 hours post infection,
there was massive proliferation of microbes in the insect with the defected gene, and this was
significantly different compared to that of the wild type.12
This data shows that bovine reproductive epithelial cells make DUOX1 and 2 mRNA
and produce DUOX1 and 2 molecules, supporting reports of ROS generation by the endometrial
epithelium in rats linked to NADPH oxidase. 14 In addition, other studies in humans indicate
that ROS are important in the menstrual cycle 32. Previous analysis of total mRNA from human
uterus, demonstrated expression of NOX11, 33 and NOX5 2, 3 which are in the same NADPH
oxidase family as DUOX. Other DUOX homologues like NOX2, NOX4, and NOX5 has been
reported to be expressed in ovaries 3.
The members of the NOX oxidase family, including DUOX, produce ROS, which
participate in the immune response in a variety of ways such as stimulating the secretion of
cytokines or the activation of other killing mechanisms, such as signaling transcription factors
like NF-κB, proteins such as kinases and phosphatases, and ion and/or proton channels. DUOX
molecules are distinct from other members of this group as they contain not only an NADPHoxidase domain, but also a domain that is homologous to heme-containing peroxidase such as
myeloperoxidase and lactoperoxidase (hence, the “dual” oxidase name). They consist of basic
NOX5 -like structure fused at the N-terminus with an additional transmembrane α-helix that is
linked at its N-terminus to the peroxidase-like domain. 6,9,18
DUOX function in the host innate mucosal response has been studied in the respiratory
epithelium and the gastrointestinal epithelium. Indeed, preliminary data from our laboratory

106

supports the importance of DUOX, by showing the expulsion of GI nematodes in rats correlated
with a 30 fold increase in DUOX expression (Mackenzie et al, manuscript in preparation)
DUOX’s role as a host innate response in the respiratory epithelium is vital for mucosal
defense against bacterial infection. Their action against bacteria is similar to that of NOX 2
acting within the phagosome, which is based on NADPH oxidase generation of O2-- followed by
dismutation to H2O2 (by phagosomal superoxide dismutase) and conversion to bactericidal
HOCl by myeloperoxidase (MPO).7,31 Other studies have shown that in the respiratory tract,
DUOX isoforms have been confirmed to be the predominant enzymes responsible for the H2O2
generation in the presence of lactoperoxidase (LPO) that is needed to catalyze a reaction between
H2O2 and thiocynate to produce the bactericidal product hypothiocynate.

11

Cytokine treatments

that increase DUOX mRNA and DUOX protein levels, induce a parallel increase in H202
production. 13
Our study results indicate that DUOX1 is more abundant in the bovine reproductive tract
than DUOX2. The role of DUOX1 and DUOX2 in both innate and adaptive host defenses of
other organs has been demonstrated by their association with either Th1 or Th2 cytokines or by
their induction through Toll-like receptors and bacterial stimuli. 13,15 Th2 cytokines have been
shown to regulated DUOX1, supporting the speculation that DUOX1 is involved in the Th2
associated clearance of T. foetus from the female reproductive tract.4 Immunohistochemistry
showed an apical localization of DUOX in the reproductive tract surface epithelia supporting the
findings from previous studies of thyroid gland and isolated cultured airway epithelial cells.5, 28
This study also indicated the presence of DUOX1and DUOX2 in bovine trophoblast cells
lines, supporting previous work by others where ROS production in human trophoblasts was
linked with an NADPH oxidase characterized as being constitutively active and distinct from the

107

phagocyte NADPH oxidase. 20, 21 These NADPH oxidases were later confirmed to be NOX2,
NOX4, and NOX5, homologues of DUOX enzymes.3,19 The physiological role of ROS
generation in the placental trophoblast has been suggested to include host defense and
degradation of noxious substances or signaling and oxygen sensing.21
The demonstration here of DUOX1 and DUOX2 presence in the bovine reproductive
tract and the endometrial cells support the hypothesis that, DUOX is expressed in the luminal
epithelium. Reactive oxygen species produced by these enzymes, play important roles in the
immune function. However, DUOX1 and DUOX2 expression in T. foetus-infected endometrial
cells were not significantly different. It is possible that in this infection model DUOX responses
either occur much faster or take longer than expected to be involved. Additional time course
studies are necessary to further investigate this. In comparison to the current study, differential
expression of DUOX1 and DUOX2 was noted when human tracheobronchial epithelial cells
were treated with inflammatory cytokines: DUOX1 increased in response to the Th 2
cytokines IL-4 and IL-13 and , DUOX2 mRNA expression increased significantly in response
to Th1 cytokine IFN-γ and IL-1a .13 DUOX1 and DUOX2 production from the T. foetus
infected bovine endometrial cell culture is consistent with the above report. However, the
DUOX1 and DUOX2 response by protozoan stimuli may be different from that of direct in
vitro stimulation by either Th1 or Th2 cytokines.
7.4 Conclusions
In this study, we investigated the presence of DUOX1 and DUOX2 in the bovine
reproductive tract by using PCR and immunohistochemistry. Results indicated the presence of
both types of DUOX: however, DUOX1 was shown to be more abundant than DUOX2 in the
reproductive tract, implying thatTh2 cytokines, associated with DUOX1, are crucial in the host

108

immune response. For future studies, we will examine more closely the DUOX response in
relation to infections in the reproductive tract in vivo. While we have here demonstrated that
neither DUOX1 nor 2 significantly change after 6 hours of T. foetus infection, and it may be that
the response occurs much faster or takes longer than expected.
The presence of DUOX molecules in our cultured endometrial and trophoblast cells
demonstrated the potential of this model to further investigate the role of these enzymes and are
particularly valuable for examining the potential role of DUOX molecules in vitro. In addition,
the presence of DUOX molecules in cows suggests that this might be a valuable species for
studying the role of innate immunity in the defense against venereal infections that cross
taxonomic lines. Our future work will utilize these valuable resources to better characterize the
DUOX response to pathogens such as. T. foetus and develop therapeutic and preventative
measures that might exploit this information.
7.5 Acknowledgements
We acknowledge the financial support of the CVM Endowment Sterner Fund. We also
acknowledge the support of Dr. Adu-Addai’s graduate committee, Drs Fitzgerald, MohanKumar, and Kaneene.

109

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A

B
Figure 7. 1. Graph showing Raw CT values for the three genes; (A).18S-EUk; (B). Bovine
Duox -1B and, (C) Bovine Duox 2B. Note. The abundance of DUOX 2 in the vagina tissue as
compare to DUOX 1.

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C
Figure 7.1 (cont’d) Graph showing Raw CT values for the three genes; (A).18S-EUk; (B).
Bovine Duox -1B and, (C) Bovine Duox 2B. Note. The abundance of DUOX 2 in the vagina
tissue as compare to DUOX 1.

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A

B

18S

C

DUOX1

DUOX2

Figure 7. 2. Amplification of the three genes in bovine reproductive tissues. (A).18S (B) . Bovine Duox -1 and, (C) Bovine Duox 2.

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Delta
Ct
values

Figure 7.3.Graph showing the Delta Ct values of DUOX1 and DUOX2 in reproductive organs
(bars represent the mean CT value from 6 animals with standard deviations). Note. Lower DCT
values indicate higher expression levels.

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.

A

B

C

D

Figure 7.4. Localization of DUOX proteins in bovine vagina and uterus by
immunohistochemistry A. Immunostaining for DUOX1 in bovine vagina. Note the heavy
DUOX1 immunostaining in the cytoplasm of the epithelium. B. Immunostaining for DUOX 1
in bovine uterus with light DUOX immunostaining in the cytoplasm of the epithelium. C.
Immunostaining for DUOX2 in bovine vagina. Note the heavy DUOX2 immunostaining in the
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Figure 7.4 (cont’d) cytoplasm of the epithelium. D. Immunostaining for DUOX2 in bovine
uterus There is less DUOX2 immunostaining in the cytoplasm of the epithelia relative to
the other sections. Immunohistochemistry for DUOX1 and DUOX2, ABC method;
counterstained with Grill 2 hematoxylin. 40x.

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A

B

C

Figure 7.5. PCR results of endometrial and trophoblast cells lines showing expression levels of 18S, DUOX1, and DUOX2. (A) The
varied expression levels of 18S for each of the cell lines. (B) Note. The expression of DUOX 1, with amplification of CT values in
the mid 30’s.indicating lower gene expression levels. (C) Note. No significant expression of either cell.

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A. Bovine endometrial cell x40

B. Bovine trophoblast x40

Figure 7. 6. Localization of DUOX1 protein in bovine endometrial and trophoblast cultured cells
by immunohistochemistry. A. Immunostaining for DUOX 1 in bovine endometrial cell. Note the
heavy DUOX immunostaining in the cytoplasm of the epithelium. B. Immunostaining for
DUOX1 in bovine trophoblast cells. Note the weaker DUOX1 immunostaining in the cytoplasm
of the trophoblast cells. Immunohistochemistry for DUOX1 with ABC method; Grill 2
hematoxylin counterstain. 40x.

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A

18S

B

18S
S

Figure 7.7. qPCR amplification of, 18S, DUOX1 (A), and DUOX2 (B) in T. foetus-infected and
uninfected bovine endometrial cells.

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Ct (normalized to 18S)

40
30
20
10
0
DUOX1 infected

DUOX1 control

DUOX 2 infected.

A

DUOX 2 Control

Ct(normalized to 18S)

40
30
20
10
0

B
Figure 7.8. DUOX1 (A) and DUOX2 (B) gene expression in cultured bovine endometrial cells.
(Bars indicate standard deviation; data is not statistically significant).

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A.

B.

C.

D.

Figure 7.9. Localization of Duox protein in bovine reproductive tissues by
immunohistochemistry. A. Immunostaining for DUOX1 in cultured T. foetus-infected bovine
endometrial cells. Heavy DUOX1 labeling is present in the cytoplasm of the cells. B.
Immunostaining for DUOX1 with lighter labeling in the cytoplasm of the uninfected endometrial
cells. C. Immunostaining for DUOX2 in infected bovine endometrial cells. Heavy DUOX2
labeling is present in the cytoplasm of the endometrial epithelium. D. Immunostaining for
DUOX2 with lighter labeling in the cytoplasm of bovine endometrial cells.
Immunohistochemistry for DUOX1 (1:200) and DUOX2 (1:25) with ABC method; Grill 2
hematoxylin counterstain. 40x.

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Chapter 8

Conclusion
Livestock reproductive diseases can have a significant effect on developing economies,
yet they are only rarely studied. Studies conducted in some affected areas in sub-Saharan Africa
showed that causative agents of such diseases are significant to livestock development and that
successful eradication of such diseases depends on epidemiological studies with the aim of
establishing mode of spread and factors that influence transmission. Additional knowledge on
the mechanisms of host immune response to these reproductive pathogens at the molecular
level including detailed information on the physical and biochemical factors involved can be
exploited for possible vaccine development.
The results of the survey using cross-sectional analysis to study some selected infectious
reproductive diseases in a Ghanaian herd of cattle indicated that T. foetus, IBR, and BHV-4 may
be possible causes for a significantly prolonged calving interval and the resulting economic
losses. Since, the report of this study is based on a single herd of cattle from a small part of
Ghana, these findings do not necessarily represent the total situation in Ghana. Better
information about these diseases is needed in order to institute appropriate treatments, as well as
prevention and control measures. To achieve this, there will be the need for a country-wide
epidemiological studies and definitive isolation of these pathogens. This study, the first to be
reported, was undertaken to provide baseline data capable of directing future studies in Ghana
with emphasis on T. foetus. The data from this study will be invaluable to Veterinary Services
Department and animal production sector in Ghana and serve as vital information for better
management of cattle.

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Experience and many clinical studies indicate that a good understanding of the
mechanism of the host innate immune response can be a key in prevention of trichomoniasis,
though investigations of the initial colonization phase have been more spars. Studies on innate
immune response during acute stage of T. foetus infection were intended to address age variation
and susceptibility to infection in a mouse model. This comparative study was done to tease out
the difference in resistance between the two age groups based on their cytokine profile. The
results showed that 4wk old mice had higher frequency of infection; however, pathological
lesions between the two showed no significance difference. There was no significance in the
uterine infection, though the 8wk groups showed a higher frequency. The 8wk old mice were
shown to be more resistant which correlated with elevation in TNF-α in both infected and the
control, implying TNF- α is constitutively secreted in the adult mice and may play a resistance
role to T. foetus infection. The Th2 cytokines (IL-2 and IL-4) in the vagina and the uterine
homogenates of the 4 wk old infected were down-regulated which were found to affect their
susceptibility compare to the rest of the group. The mechanism leading to this down-regulation is
not understood and needs to be further investigated. Contrary to the earlier reports from various
studies that T. foetus infection is primarily handled by Th2 pathway cytokines, the results from
our study indicated that both Th1 and the Th2 pathways seem to be involved in the response to T.
foetus infection. However, TNF-α has been variably associated with Th1, Th2, or neither;
instead it appears to be proinflammatory, participating in either Th milieu.
DUOX1 and DUOX2, members of the NADPH oxidase family, function in the mucosal
surface innate host response. When human tracheobronchial epithelial cells were treated with
inflammatory cytokines, DUOX1 increased in response to the Th 2 cytokines (IL-4) and DUOX2

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mRNA expression increased significantly in response to Th1 cytokine interferon gamma (IFN-γ),
implying differential expression.
The goal of Chapter 7 study was to confirm such differential expression in bovine
endometrial cells infected 6hrs with an extracellular protozoan (T. foetus), known to respond to
Th2 response. These were done in three steps; first, we documented the presence of DUOX in
the reproductive tract of bovine (in vivo). Second, we confirmed DUOX1 and DUOX2 were
present in the bovine endometrial and trophoblast cell lines, but not in T. foetus or in the media.
Finally, we infected endometrial cell lines with T. foetus for 6hrs to test whether DUOX1 or
DUOX2would significantly increase in response to the acute stage of T. foetus infection. The
results demonstrate that neither DUOX1 nor 2 significantly change after 6 hours of T. foetus
infection. This implies that if there is a response, it must either occur faster or take longer than
expected. In summary, the study did not confirm nor deny the differential expression of DUOX1
and DUOX2 in the reproductive tract mucosal surface; however, it demonstrated that like airway
and thyroid epithelial cells, reproductive tract mucosal surface also expressed DUOX1 and
DUOX2.
Based on these results, our future studies will focus on additional time course studies
from 0-48hrs at 2 hr intervals determining DUOX expression during infection. Also, different
models for both in vivo and in vitro studies will be considered to maximize the chance of
DUOX expression. In addition, the mechanism of DUOX innate immune protection in the
reproductive tract will be further elucidated. The presence of DUOX molecules in our cultured
endometrial cells makes this a particularly valuable model for examining the potential role of
DUOX molecules in vitro.

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Our future work will utilize this valuable model to better characterize the DUOX
response to pathogens such as. T. foetus and develop therapeutic and preventative measures that
might exploit this information.

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