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Title Studies on Improvement and Validation of Dembo-PCR forDetection of Domestic Animal Pathogens( 本文(Fulltext) )
Author(s) Sayed Samim RAHPAYA
Report No.(DoctoralDegree) 博士(獣医学) 甲第513号
Issue Date 2018-09-21
Type 博士論文
Version ETD
URL http://hdl.handle.net/20.500.12099/77274
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Studies on Improvement and Validation of Dembo-PCR
for Detection of Domestic Animal Pathogens
Dembo-PCR
2018
The United Graduate School of Veterinary Sciences
Gifu University
(Tokyo University of Agriculture and Technology)
Sayed Samim RAHPAYA
a
Abbreviations ............................................................................................................................. i
General Introduction ................................................................................................................ 1
A. Abortion ............................................................................................................................. 3
A.1. Bluetongue virus ......................................................................................................... 4
A.2. Akabane virus ............................................................................................................. 4
A.3. Simbu group viruses .................................................................................................... 4
A.4. Ibaraki virus ................................................................................................................ 5
A.5. Chuzan virus ............................................................................................................... 5
A.6. Chlamydophila abortus ............................................................................................... 5
A.7. Brucella abortus .......................................................................................................... 6
A.8. Campylobacter fetus ................................................................................................... 6
A.9. Listeria monocytogenes ............................................................................................... 7
A.10. Leptospira spp. .......................................................................................................... 7
A.11. Toxoplasma gondii .................................................................................................... 7
A.12. Neospora caninum .................................................................................................... 7
A.13. Sarcocystis spp. ......................................................................................................... 8
A.14. Aspergillus spp. ......................................................................................................... 8
B. Diarrhea .............................................................................................................................. 8
B.1. Bovine enterovirus ...................................................................................................... 9
B.2. Rotavirus ..................................................................................................................... 9
B.3. Bovine torovirus .......................................................................................................... 9
B.4. Mammalian orthoreovirus ......................................................................................... 10
B.5. Bovine leukemia virus ............................................................................................... 10
B.6. Mycobacterium avium subspecies paratuberculosis .................................................. 10
B.7. Clostridium perfringens ............................................................................................ 10
B.8. Escherichia coli ......................................................................................................... 11
B.9. Eimeria bovis/Eimeria bovis ..................................................................................... 11
C. Respiratory disease complex ............................................................................................ 11
Table of contents
b
C.1. Bovine parainfluenza virus 3 ..................................................................................... 12
C.2. Bovine respiratory syncytial virus ............................................................................. 12
C.3. Influenza D virus ....................................................................................................... 13
C.4. Bovine rhinitis virus .................................................................................................. 13
C.5. Mannheimia haemolytica .......................................................................................... 13
C.6. Histophilus somni ...................................................................................................... 13
C.7. Trueperella pyogenes ................................................................................................ 14
C.8. Mycobacterium bovis ................................................................................................ 14
C.9. Mycoplasma bovis ..................................................................................................... 14
D. Multi-diseases pathogens ................................................................................................. 15
D.1. Bovine viral diarrhea virus ........................................................................................ 15
D.2. Bovine herpesvirus-1 ................................................................................................ 15
D.3. Bovine coronavirus ................................................................................................... 15
D.4. Bovine adenovirus ..................................................................................................... 16
D.5. Salmonella spp. ......................................................................................................... 16
E. Transmission of infectious diseases ................................................................................. 16
E.1. Vectors ....................................................................................................................... 16
E.2. Reservoirs .................................................................................................................. 17
F. Detection system of microbes for bovine diseases by real-time PCR .............................. 17
CHAPTER ONE. Development of Dembo-PCR for Detection of Infectious Agents of Abortion (Dembo abortion-PCR) .......................................................................................... 19
I.1. Introduction .................................................................................................................... 20
I.2. Materials and methods .................................................................................................... 21
I.2.1. Primers and probes design ....................................................................................... 21
I.2.2. Preparation of genomic DNA and RNA ................................................................ 22
I.2.3. Real-time PCR amplification ................................................................................... 22
I.2.4. Sensitivity test .......................................................................................................... 22
I.2.5. Specificity test ......................................................................................................... 23
I.2.6. Clinical samples ........................................................................................................... 23
I.2.6.1. Blood samples ....................................................................................................... 23
I.2.6.2. Aborted fetus samples .......................................................................................... 23
c
I.3. Results ............................................................................................................................ 23
I.3.1. Sensitivity and of the Dembo abortion-PCR ........................................................... 23
I.3.2. Specificity of Dembo abortion-PCR ........................................................................ 24
I.3.3. Clinical samples ...................................................................................................... 24
I.3.3.1. Blood samples .................................................................................................. 24
I.3.3.2. Aborted fetus samples ...................................................................................... 24
I.4. Discussion ....................................................................................................................... 24
CHAPTER TWO. Detection of Infectious Agents of Abortion, and Respiratory Disease Complex in Potential Vectors and Reservoirs with Dembo-PCR ....................................... 43
II.1. Introduction ................................................................................................................... 44
II.2. Materials and methods .................................................................................................. 45
II.2.1. Primer and probe design ......................................................................................... 45
II.2.2. Extraction of nucleic acids ..................................................................................... 45
II.2.3. Real-time PCR amplification ................................................................................. 46
II.2.4. Analysis of field samples ....................................................................................... 46
II.3. Results ........................................................................................................................... 47
II.3.1. Sensitivity and specificity of the Dembo abortion-PCR ........................................ 47
II.3.2. Analysis of field samples by Dembo-PCR ............................................................. 47
II.4. Discussion ..................................................................................................................... 47
CHAPTER THREE. Screening of Nasal and Fecal Samples from Goats for Detection of Infectious Agents of Abortion, Diarrhea, and Respiratory Disease Complex by Dembo-PCR ......................................................................................................................................... .62
III.1. Introduction: ................................................................................................................. 63
III.2. Material and methods ................................................................................................... 64
III.1.1. Primers and probes ................................................................................................ 64
III.1.2. Extraction of nucleic acids .................................................................................... 64
III.1.3. Real-time PCR amplification ................................................................................ 65
III.1.4. Clinical samples .................................................................................................... 65
III.2. Results: ......................................................................................................................... 66
III.2.1. Analysis of clinical samples by using Dembo-PCR ............................................. 66
III.3. Discussion .................................................................................................................... 66
General Conclusion ................................................................................................................. 79
d
Acknowledgements ................................................................................................................. 82
References ................................................................................................................................ 85
i
Abbreviations
AKAV Akabane virus
BAdV Bovine adenovirus
BCoV Bovine coronaviruses
BEV Bovine enterovirus
BHV-1 Bovine herpesvirus-1
BLV Bovine leukemia virus
BRAV Bovine rhinitis A virus
BRBV Bovine rhinitis B virus
BRDC Bovine respiratory disease complex
BRSV bovine respiratory syncytial virus
BRV Bovine rhinitis viruses
BT Bluetongue
BTV Bluetongue virus
BVD Bovine viral diarrhea
BVDV Bovine viral diarrhea virus
cDNA complementary DNA
CHUV Chuzan virus
Dembo-PCR Detection system of microbes for bovine diseases by real-time
PCR
ii
dsRNA Double-stranded RNA
E PCR efficiency
E. coli Escherichia coli
EHDV Epizootic hemorrhagic disease virus
EHDV-2 Epizootic hemorrhagic disease virus, serotype 2
ERV Equine rhinitis virus
FAM 6-carboxyfluorecein
FMDV Foot and mouth disease virus
IBAV Ibaraki virus
IDV Influenza D virus
LOD limit of detection
MRV Mammalian orthoreovirus
PIV3 Parainfluenza virus type 3
R2 Correlation coefficient
T1L Type 1 Lang
T2J Type 2 Jones
T3A Type 3 Abney
T3D Type 3 Dearing
TAMRA 6-carboxytetramethylrhodamine
USDA United States Department of Agriculture
1
«General Introduction»
2
Abortion, diarrhea and respiratory disease complex are the important diseases in
cattle, and these major health problems, induce high and significant economic loses for
the cattle industry [13, 28, 69, 107]. In the United States (US), late-term cattle abortions
have been calculated to cost between $500 and $900 per case [107], and bovine
respiratory disease complex is estimated to account for 70–80% of all feedlot cattle
morbidity and 40–50% of all cattle mortality, resulting in a loss of greater than the US
$500 million per year [71].
The infectious agents are one of the important causative agents in these diseases
[13, 28, 107]. The pathogens could be transmitted in various ways including physical
contact between animals, consumption of food, body fluids, airborne inhalation, or
through vectors and reservoirs [9, 13, 33, 54, 114]. Vectors and reservoirs such as
rodents, birds, arthropods, insects including flies and even other animals including
goats, dogs, and cats may play important roles in transmission and reservation of cattle
infectious agents [13, 14, 23, 33, 54, 114].
Serological tests, pathogen isolation, and PCR-based tests are currently available
to diagnose bovine abortion, diarrhea, and respiratory disease complex infectious agents
in laboratories, however; most tests are targeting one pathogen, which shows lack of
systems for simultaneous detection of pathogens [77, 86].
To address these problems, development of a simpler, faster, more
comprehensive, accurate, and rapid system for detection and differentiation of bovine
abortogenic, diarrheal and respiratory diseases infectious agents is essential. For this
purpose, two types of detection system of microbes for bovine diseases by real-time
PCR (Dembo-PCR), Dembo-PCR for diarrheal including 19 pathogens (Dembo
diarrhea-PCR) and for respiratory disease complex including 16 pathogens (Dembo
respiratory-PCR) were already developed [86, 146].
3
In this study, I developed a Dembo-PCR for abortion including 24 agents
(Dembo abortion-PCR) and evaluated whether the infectious agents in cattle were
transmitted by vectors and reservoirs, and goats. This system has high sensitivity, high
specificity, and rapidity, and it is able to detect all target pathogens in single-run
simultaneously, within 3 hours.
This thesis was constructed with three chapters as follow;
Chapter one. Development of Dembo-PCR for detection of infectious agents of
abortion (Dembo abortion-PCR), Chapter two. Detection of infectious agents of
abortion, and respiratory disease complex in potential vectors and reservoirs with
Dembo-PCR, and Chapter three. Screening of nasal and fecal samples from goats for
detection of infectious agents of abortion, diarrhea, and respiratory disease complex by
Dembo-PCR.
In Chapter one, I established a detection system of microbes for bovine abortion
diseases by real-time PCR (Dembo abortion-PCR), a newly developed detection system
for 24 bovine abortogenic agents.
In Chapter two, I evaluated whether the infectious agents causing diseases in
cattle, could be transmitted by various kinds of vectors and reservoirs such as insects,
rodents, arthropods, and birds by using the Dembo-PCR systems.
In Chapter three, I evaluated whether the infectious agents causing abortion,
diarrhea, and respiratory disease complex in cattle, could be detected in goats by using
the Dembo-PCR systems.
A. Abortion
Abortion is the delivery of an immature fetus, either live or dead, before the
completion of the gestation period [9, 13, 29]. Abortion in cattle could occur due to
infectious and non-infectious causes. The infectious abortion is caused by bacteria,
viruses, protozoa and fungi, and the non-infectious abortion is related to same factors,
4
including toxins, hormonal agents, physical and nutritional factors [9, 54]. The most
common infectious agents of cattle abortion are as follow;
A.1. Bluetongue virus
Bluetongue (BT) is an infectious and arthropod-borne viral disease of ruminants
and camelids. BT virus (BTV) belongs to the genus Orbivirus in the family Reoviridae
and has 26 serotypes. BTV transmits by biting midges, genus Culicoides, family
Ceratopogonidae. BTV is widely distributed in the tropics and subtropics due to the
existence of Culicoides species [49, 163]. BT Develop with high fever,
salivation, swelling of the face and cyanosis of the tongue, resulting in mortality,
mortality, and abortion in sheep and occasionally in goats and cattle [49, 124, 163].
A.2. Akabane virus
Akabane virus (AKAV) is an arthropod-borne virus. It belongs to the
genus Orthobunyavirus of the family Peribunyaviridae (Order Bunyavirales), which
characterized by a tripartite negative-sense RNA genome with large (L), medium (M),
and small (S) segments [75, 140]. It was first isolated in Japan in 1959 [75]. It should
be noted that the virus is one of the critical agents of epizootic abortions, stillbirths and
congenital abnormalities of ruminants in Asia, the Middle East, and Australia. AKAV
was initially isolated from mosquitoes, but Culicoides biting midges are thought to be
its primary vectors [88].
A.3. Simbu group viruses
Simbu group viruses belong to the genus Orthobunyavirus of the
family Peribunyaviridae (Order Bunyavirales) and contain over 179 viruses, which are
essential for medical and veterinary fields [101]. These viruses are transmitted by
mosquitoes and Culicoides biting midges. At least 25 out of 179 viruses of
genus Orthobunyavirus belongs to the Simbu group and divided into seven species
including Manzanilla virus, Oropouche virus, Sathuperi virus, Shamonda virus, Shuni
5
virus, Aino virus and Simbu virus [101]. Most of the members of this genus cause sub-
clinical infections in non-pregnant animals [158]. In pregnant animals, some of these
viruses readily cross the placenta resulting in fetal infections that are associated with
abortion, premature birth, stillbirth and congenital abnormalities in calves and lamb
[101, 158].
A.4. Ibaraki virus
Ibaraki virus (IBAV) is a strain of the epizootic hemorrhagic disease virus
(EHDV) serogroup, which belongs to the genus Orbivirus, family Reoviridae. IBAV
cross-reacts serologically with EHDV serotype 2 (EHDV-2), thus; the virus is now
classified as a strain of EHDV-2 [20]. IBAV is an arthropod-borne virus and transmitted
by blood-feeding midges, Culicoides spp., and affects wild and domestic ruminants
[102]. Ibaraki disease of cattle caused by IBAV was first reported, as a disease with
fever, anorexia, salivation, deglutition disorder and occasionally causes abortion [111].
After the first case, a few outbreaks in cattle in Turkey, Morocco, the French island of
Réunion and Israel, with high morbidity and mortality have been reported [102, 133,
157].
A.5. Chuzan virus
Chuzan virus (CHUV) is one of the members of Palyam serogroup, which is
related to the genus Orbivirus in the family Reoviridae. It causes congenital diseases
including abortion and infertility in cattle [3]. For the first time, CHUV was identified
from a biting midge (Culicoides oxystoma) and sentinel calves in 1985 in Japan [3].
A.6. Chlamydophila abortus
Chlamydophila abortus is a Gram-negative obligate intra-cellular bacterium
[76]. It is one of the common causes of abortion in cattle [44, 124]. The family
Chlamydiaceae divided into the genera Chlamydophila and Chlamydia, which have a
total of 9 species, namely, Chlamydophila abortus, C. pecorum, C. psittaci, C.
6
pneumoniae, C. felis, C. caviae, Chlamydia trachomatis, C. suis and C. muridarum.
Three new families were added, i.e., Parachlamydiaceae, Waddliaceae, and
Simkaniaceae [44].
C. abortus causes various diseases in animals and humans. In cattle, C. abortus
cause abortion, infertility, polyarthritis, encephalomyelitis, keratoconjunctivitis,
pneumonia, enteritis, hepatitis, vaginitis and endometritis, and chronic mastitis [15, 34,
81, 112, 115].
A.7. Brucella abortus
Bacteria of the genus Brucella are Gram-negative, non-encapsulated and
facultative intracellular coccobacillus [63, 128]. The genus Brucella has six species
including Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis and B. neotomae
[63].
B. abortus one of the critical cause of abortion and infertility in cattle and is one
of the crucial pathogens of zoonoses, which is distributed worldwide [124, 128].
A.8. Campylobacter fetus
Campylobacter fetus is a species of genus Campylobacter, which is a Gram-
negative, curved or spiral, polar flagellated, microaerophilic bacterium [72].
Campylobacteriosis is a venereal disease of cattle and sheep caused by the
bacteria C. fetus subsp. fetus, which was formerly known as Vibrio
fetus subspecies venerealis. The disease causes infertility in the cattle. However,
abortion in late gestation could occasionally be observed. Most cases or outbreaks occur
after the recent introduction of an infected bull or cow into a susceptible breeding herd
[72, 124, 144].
7
A.9. Listeria monocytogenes
Listeria monocytogenes is a Gram-positive, facultative anaerobic and rod-
shaped bacterium of genus Listeria. The genus Listeria contains 10 species including L.
fleischmannii, L. grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L.
rocourtiae, L. seeligeri, L. weihenstephanensis and L. welshimeri. L. denitrificans [25].
L. monocytogenes is one of the causative agents of abortion and infertility in cattle [108,
124].
A.10. Leptospira spp.
Leptospira is a member of the family Leptospiraceae, one of the causative
agents of abortion in cattle [124, 136].
Leptospirosis is a zoonosis with distribution through the world, which is caused
by Leptospira interrogans. Leptospirosis causes significant economic loss to the cattle
industry. L. interrogans serovar Hardjo type Hardjobovis is the primary cause of acute
and chronic leptospirosis in cattle, and also causes persistent infection in the female
reproductive tract of infected cattle, resulting in infertility and abortion [136].
A.11. Toxoplasma gondii
Toxoplasma gondii is a zoonotic protozoan parasite. It is one of the most
pathogenic infectious agent of cattle and other animals [123]. The sexual phase of
protozoan develops in the intestine of cats as definitive hosts, and other animals such as
cattle are the intermediate hosts of the parasite [43]. It is one of the foremost cause of
abortion in animals including cattle [124].
A.12. Neospora caninum
Neospora caninum is an obligate intracellular coccidian parasite with a
worldwide range of distribution. It is one of the critical cause of abortion in cattle [39].
N. caninum can infect a broad spectrum of wild and domestic animals. Dog, coyote and
gray wolf (Canis lupus) are considered to be the final host of N. caninum [38]. While
8
mammals and birds, including cattle, sheep, goat, water buffalo, horse, donkey, bison,
white-tailed deer, Red fox, chicken, pigeon, sparrow, feral swine, capybara, and rabbit
can be potential natural intermediate hosts of this pathogen [39, 134]. Although N.
caninum has been detected in several mammals and birds, lifecycle and hosts of this
pathogen are not fully understood [124].
A.13. Sarcocystis spp.
Sarcocystis spp. are cyst-forming coccidian parasites of family Apicomplexa,
and they have obligatory two-host life cycle including carnivorous as definitive hosts
and herbivorous or omnivorous as intermediate hosts [46]. Sarcocystis spp. cause
sarcocystosis in domestic animals and each intermediate and a definitive host may be
infected with more than one Sarcocystis species [6]. Cattle and water buffaloes are the
intermediate hosts of the critical species of Sarcocystis including Sarcocystis cruzi and
Sarcocysits hirsuta, which cause Sarcocystosis and occasionally abortion [55].
Intermediate hosts become infected with the parasite via ingesting sporocysts or
sometimes sporulated oocysts existed in the food or water [37].
A.14. Aspergillus spp.
Aspergillus spp. belonging to the family Trichocomaceae, are saprophytic
filamentous fungi that are commonly found in various climates worldwide, and can
infect living hosts including plants, insects, birds, and mammals [120]. Aspergillus spp.
are one of the causative agents of bovine mycotic abortion [124].
B. Diarrhea
Diarrhea in cattle causes significant economic loss by decreasing fertility and
productivity, including milk production and weight gain [69]. Young calves strongly
suffer from diarrhea which sometimes resulted in death by malnutrition and dehydration
[24]. According to the reports of the United States Department of Agriculture (USDA),
57% of deaths of weaning calves in the US were caused by diarrhea [146]. Even though
9
the cattle industry has taken measures to prevent diarrhea, such as hygiene and feeding
management, it still stays as a major worldwide problem for cattle industry [24, 69].
The most common infectious agents of cattle diarrhea are as follow;
B.1. Bovine enterovirus
Bovine enterovirus (BEV) is a single-stranded RNA virus belonging to the genus
Enterovirus a member of the family Picornaviridae. Enteroviruses are widely
distributed viruses, which infect a broad spectrum of mammals. Eighty-nine serotypes
of enterovirus have been identified: 62 related to human infections and 27 associated
with animal infections [82, 147]. BEV is expected to be endemic in cattle in many
regions [7]. BEV is known to infect subclinically in healthy animals. However, BEV
could be associated with diarrhea, abortions and respiratory disease [86, 146].
B.2. Rotavirus
Rotaviruses are members of the genus Rotavirus in the family Reoviridae,
whose members could infect various host species including mammals, reptiles, fish,
birds, fungi, plants and insects [67, 79]. Rotaviruses are classified into 8 Groups (A to
H) based on the genetic property of their inner capsid protein VP6 [103]. Group A
rotaviruses and Group B rotaviruses cause diarrhea in animals including cattle [146].
B.3. Bovine torovirus
Torovirus is a single-stranded, enveloped and positive-sense RNA virus in the
family Coronaviridae, order Nidovirales [61]. Toroviruses cause gastroenteritis and
diarrhea in mammals including humans, horses, cattle, and swine worldwide [61, 146].
Bovine torovirus was first identified in the US during an outbreak of diarrhea in cattle
in 1979 [156]. Bovine torovirus is one of the most critical diarrheal infectious agents in
cattle [89].
10
B.4. Mammalian orthoreovirus
Reoviridae contains 2 subfamilies: Spinareovirinae and Sedoreovirinae,
including 9 and 6 genera, respectively, which are non-enveloped viruses with a
segmented genome of 10 to 12 double-stranded RNA (dsRNA) segments [30]. They
could infect various host species including mammals, reptiles, fish, birds, protozoa,
fungi, plants, and insects [30, 92]. The mammalian orthoreovirus (MRV) is one of the
species of Reoviridae. It is divided into 3 serotypes based on neutralization of
infectivity, and hemagglutination inhibition by type-specific antisera, with the prototype
isolates being type 1 Lang (T1L), type 2 Jones (T2J), type 3 Dearing (T3D) and Abney
(T3A). MRV causes respiratory, enteric diseases and diarrhea in animals including
cattle [86, 146].
B.5. Bovine leukemia virus
Bovine leukemia virus (BLV) is a retrovirus that infects cattle [129]. The
majority of animals infected with BLV are asymptomatic carriers of the virus. BLV
infects cattle via horizontal transmission, mainly through the transfer of infected cells
[19]. BLV is a worldwide distributed virus. In the US, 38% of beef herds, 84% of all
dairy herds, and 100% of large-scale dairy operation herds are infected with BLV [2].
B.6. Mycobacterium avium subspecies paratuberculosis
Mycobacterium avium subspecies paratuberculosis is a Gram-positive, obligate
pathogenic bacterium of genus Mycobacterium [148]. It can infect the gastrointestinal
tract of a range of hosts including cattle and is the known cause of Johne’s disease in
ruminants, a disease characterized by diarrhea, weight loss and eventual death [141,
145].
B.7. Clostridium perfringens
Clostridium perfringens is a Gram-positive, ubiquitous, spore-forming, soil-
borne bacterium. Six toxigenic types of the organism including A, B, C, D, E, and F are
11
identified [151]. All types except F have been implicated in diseases of cattle [151]. It
is commonly found in several environments such as soil, water, poorly preserved feeds,
contaminated or improperly thawed colostrum or milk, calf housing environments, and
the normal bovine intestinal tract. Usually, C. perfringens is innocuous in the intestine,
but under the right conditions they (specially type A) grow and proliferate, resulting in
enterotoxaemia and diarrhea [135, 146].
B.8. Escherichia coli
Escherichia coli is a Gram-negative, facultative anaerobic, rod-shaped
bacterium of the genus Escherichia [96]. E. coli was first described by Theodor
Escherich in 1885. Most strains of E. coli are harmless and live as normal flora in the
gastrointestinal tract of humans and animals. However, some strains could be
pathogenic by acquiring virulence factors through plasmids, transposons, and
bacteriophages [53, 96].
Cattle are one of the reservoirs of the pathogenic bacteria E. coli, and
approximately 30% of feedlot cattle shed E. coli [53, 146].
B.9. Eimeria bovis/Eimeria bovis
Among 20 spices of Eimeria spp. in cattle, Eimeria bovis and E. zuernii are the
most pathogenic Eimeria species causing cattle coccidiosis [29, 64].
The prevalence of Eimeria spp. in cattle is high, which almost reach 100% in
calves within three weeks to 6 months [29], and commonly it causes diarrhea [146].
C. Respiratory disease complex
Bovine respiratory disease complex (BRDC) is one of the most important
diseases for the cattle industry, due to significant economic damages including
mortality, morbidity, treatment costs and feed inefficiency [71, 73, 86]. For example,
BRDC is calculated to be involved in 70–80% of all feedlot cattle morbidity and 40–
12
50% of all cattle mortality, resulting in economic loss of greater than US $500 million
per year in the US [86, 107].
The most common infectious agents of cattle respiratory disease complex are as
follow:
C.1. Bovine parainfluenza virus 3
Parainfluenza virus type 3 (PIV3) is an enveloped, and single-stranded negative-
sense RNA virus belongs to the Respirovirus genus of the Paramyxoviridae family [90,
100].
Bovine PIV3 (BPIV3) is one of the BRDC agents, and it is one of the most
significant causes of diseases in feedlot cattle worldwide. However, other respiratory
viruses such as bovine herpesvirus 1 (BHV-1), bovine viral diarrhea virus (BVDV) and
bovine respiratory syncytial virus (BRSV) have also been related to the BRDC
development in feedlot cattle [86, 90, 100]. Clinically, the symptoms in the infected
cattle with BPIV3 can be different, ranging from asymptomatic infections to severe
respiratory illness. In usual cases, the infected cattle with BPIV3, shows the clinical
signs such as coughing, fever and nasal discharge [100].
C.2. Bovine respiratory syncytial virus
Bovine respiratory syncytial virus (BRSV) is a single-stranded, negative-sense
RNA virus. It belongs to the genus Pneumovirus in the subfamily Pneumovirinae,
family Paramyxoviridae [8]. BRSV is a significant cause of respiratory disease
worldwide [86, 121, 131]; thus disease caused by BRSV is a major part of BRDC.
BRSV could infect the upper and lower respiratory tract and is shed in nasal discharges
[131].
13
C.3. Influenza D virus
Influenza D virus (IDV) is a single-stranded, negative-sense RNA virus of the
family Orthomyxoviridae [50]. IDV was first isolated from swine with respiratory
disease in 2011 [66], After that, the identification of IDV in cattle was reported from
several areas including the US, France, and China. Subsequently, the virus was isolated
from sheep and goats [50]. IVD plays an essential role as a trigger of BRDC [86].
C.4. Bovine rhinitis virus
Bovine rhinitis A virus (BRAV) and Bovine Rhinitis B virus (BRBV) are
members of the genus Aphthovirus, family Picornaviridae, along with equine rhinitis
virus (ERV) and foot and mouth disease virus (FMDV) [71]. Two serotypes of BRAV
have been identified, BRAV1 and BRAV2, while BRBV consists of a single serotype
[65]. Bovine rhinitis viruses (BRV) are the important etiological agents of bovine
BRDC [86].
C.5. Mannheimia haemolytica
Mannheimia haemolytica is a Gram-negative, anaerobic, non-spore-forming
species of genus Mannheimia in the family Pasteurellaceae [126, 160]. It is the one of
important part of the BRDC, and common bacterium isolated from respiratory disease
in cattle, especially, in feedlot cattle and is a significant member of enzootic pneumonia
in all neonatal calves [1, 126]. M. haemolytica plays an opportunistic role by passing
the lungs when stress or infection compromise host defenses with respiratory viruses or
mycoplasma [86, 160].
C.6. Histophilus somni
Histophilus somni is a Gram-negative, opportunistic and facultative pathogenic
bacterium [11, 118]. It was formerly named as Histophilus ovis, Haemophilus agni and
Haemophilus somnus [11]. For the first time, Histophilus somni was isolated in
Colorado, USA, in 1956, as an infectious agent of encephalitis in cattle [18],
14
subsequently, as causing meningoencephalitis and thromboembolic
meningoencephalomyelitis [62], pneumonia [10], otitis [36], and mastitis [70]. In sheep,
H. somni causes orchitis, epididymitis, and mastitis [116], abortion [105], synovitis,
meningoencephalitis [119], septicemia, and pneumonia [86]. There are only two reports
of identification of H. somni in healthy goats [118], however; there is no information
about clinical infection by this pathogen in goats, but goats could be potential reservoirs
H. somni for cattle and sheep.
C.7. Trueperella pyogenes
Trueperella pyogenes (first named Aeranobacterium pyogenes) is a Gram-
positive, commensal and opportunistic pathogenic bacterium of genus Trueperella,
causing several diseases, such as mastitis, liver abscessation, and pneumonia [162]. It
is a commensal pathogen of the oropharynx, upper respiratory, and gastrointestinal
tracts of livestock [86, 127, 162]. It may be transmitted mechanically by biting flies
and dairy equipment [127].
C.8. Mycobacterium bovis
Mycobacterium bovis is a Gram-positive, slow-growing aerobic bacterium of the
genus Mycobacterium [26, 31]. It causes tuberculosis in cattle worldwide and is one of
the major animal and public health problem in the countries in which people are in close
contact with their cattle [31, 146]. Bovine tuberculosis may be transmitted by cattle-to-
cattle contact and also through the involvement of wildlife reservoirs [31, 124].
C.9. Mycoplasma bovis
Mycoplasma bovis is one of the smallest cell-wall deficient bacterium in the
genus Mycoplasma [21]. It is a pathogen causing respiratory disease, otitis media,
arthritis, mastitis, and a variety of other conditions in cattle worldwide [104], and it is
one of the impartment members of BRCD [86].
15
D. Multi-diseases pathogens
There are several pathogens which have potential to causes diarrhea, abortion
and respiratory disease in cattle, simultaneously [86, 124, 146]. The following
pathogens are infectious agents of abortion, diarrhea, and respiratory disease complex;
D.1. Bovine viral diarrhea virus
Bovine viral diarrhea (BVD) is a viral disease of cattle and other ruminants that
is caused by the BVD virus (BVDV), which belongs to the genus Pestivirus in the
family Flaviviridae. BVDV is one of the important causative agents of abortion,
diarrhea and respiratory diseases in cattle [44, 52, 69, 86, 124, 146]. BVDV causes
BVD, which was first reported as a transmissible disease in 1946 and is a critical cause
of respiratory and reproductive disorders in cattle [45, 52, 69]. After that, BVD has been
reported worldwide [17]. Two genotypes, BVDV type 1 and BVDV type 2, are further
classified as cytopathogenic or non-cytopathogenic based on in vitro cell culture
characteristics [45].
D.2. Bovine herpesvirus-1
Bovine herpesvirus-1 (BHV-1) is an enveloped, double-stranded DNA virus
belonging to the family Herpesviridae [60]. It causes infectious bovine rhinotracheitis,
a highly contagious respiratory disease of cattle [60]. BHV-1 is associated with clinical
syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis,
abortion, infertility, conjunctivitis and encephalitis and diarrhea [60, 86, 146]. The main
route of transmission is via nasal exudates and the respiratory droplets, genital
secretions, semen, fetal fluids and tissues [60, 86].
D.3. Bovine coronavirus
Bovine coronaviruses (BCoV) belong to the family Coronaviridae in the
order Nidovirales and are classified in subgroup 2a in coronaviruses [132]. They cause
diarrhea and respiratory disease in cattle and wild ruminants [86, 132, 146]; thus, BCoV
16
is a pneumoenteric virus with the ability to infect both part or respiratory system (upper
and lower respiratory tract) and intestine [86, 146].
D.4. Bovine adenovirus
Bovine adenovirus (BAdV) is a double-stranded DNA virus, belonging to the
family Adenoviridae [91, 138]. BAdV has 10 serotypes, which are distributed
worldwide [138]. BAdV, particularly, BAdV serotypes 3 and 7 play an important role
in cattle respiratory illness as a part of BRDC [86], and cattle diarrhea [146].
D.5. Salmonella spp.
Salmonella is a Gram-negative pathogenic bacterium genus of the family
Enterobacteriaceae [106]. It has 2 species including Salmonella enterica and
Salmonella bongori [106, 153]. Salmonella enterica ser. enterica, S. enterica ser.
Typhimurium and S. enterica ser. Dublin causes diarrhea and abortion in animals
including cattle [124, 146].
E. Transmission of infectious diseases
Transmission of pathogens could be occurring in various ways including
physical contact between animals, consumption of food, body fluids, airborne
inhalation, and through vectors and reservoirs [149].
E.1. Vectors
Vectors including insects, such as mosquitoes, flies, ticks, fleas, and lice play
important roles in transmission of the pathogens [12, 32, 33].
Generally, vectors are divided into 2 types, biological vectors and mechanical
vectors [59]. Biological vectors transmit infectious agents within their bodies, where the
infectious agents undergo multiplication and/or development, consequently transmitting
the infectious agents to the host through bites. Mosquitoes are a biological vector of
many pathogens [124]. Mechanical vectors transfer pathogens from an infected host or
17
a contaminated substrate to a susceptible host without multiplying and/or developing of
the pathogens within the vector. Many insects can serve as mechanical vectors. Many
insects are the examples of mechanical vectors [59, 124].
E.2. Reservoirs
Reservoir is one or more epidemiologically connected populations or
environments in which the infectious agent can be persistently maintained and from
which infectious disease is transmitted to the defined target population. One pathogen
may cause disease in multiple target populations, and the reservoir for each target
population can be different. Most known disease reservoirs are mammals such as
rodents and carnivores [68].
F. Detection system of microbes for bovine diseases by real-time PCR (Dembo-
PCR)
Dembo-PCR is a TaqMan real-time PCR-based assay to detect multiple
pathogens simultaneously with high sensitivity, high specificity, rapidity. In Dembo-
PCR, a broad range of pathogens could be detected simultaneously in a panel [145].
As Dembo-PCR workflow, extracted DNA and RNA (template), from
specimens, reagents and specific primer-set and probe of each pathogen were mixed in
individual reaction tubes. A LightCycler Nano and the Applied Biosystems 7300 Real-
Time PCR instruments were used for all qPCR reactions (Fig. A.1).
Dembo-PCR was developed in 3 steps. Dembo-diarrhea-PCR for detection of
bovine diarrheal agents [146], Dembo-respiratory-PCR for detection of bovine
respiratory disease complex [86], and Dembo abortion-PCR for detection of bovine
abortive agents [124]. Subsequently, it was evaluated whether infectious agents causing
diarrhea, respiratory disease complex and abortion in cattle were transmitted by vectors
and reservoirs such as flies, arthropods, rodents, birds, and other animals such as goats,
with Dembo-PCR system [124].
18
Fig. A.1. Dembo-PCR workflow.
19
CHAPTER ONE
« Development of Dembo-PCR for Detection of Infectious Agents of Abortion
(Dembo abortion-PCR)»
20
I.1. Introduction
Bovine abortion is the expulsion or production of a dead calf between 42 and
260 days of gestation [13, 42, 47]. Abortion is the vast majority of reproductive failures
in cattle herds, with putting highly negative impacts and facing the cattle industry with
significant and considerable economic losses [9, 28, 54].
Bovine abortion is occurred due to infectious and non-infectious causes. The
infectious abortion is caused by bacteria, viruses, protozoa, and fungus, and the non-
infectious abortion is related with same factors, including toxins, hormonal agents,
physical and nutritional factors [110, 124]. One report in the US shows, that 18%,
14.6%, 3.2% and 1.3% of infectious agents associated with bovine abortion were
bacteria, protozoa, fungi and viruses, respectively, with 5.5% due to non-infectious
causes and 57.3% undetermined [78]. However, in other findings, bacteria and fungi
were the major causative agents of infectious abortions, but in the recent years, several
researchers show, that almost 30% of all abortions in North America and Europe were
associated with N. caninum [86].
Abortion by infectious agents are almost with same symptoms, and it is difficult
to diagnose it according to the clinical signs without exact pathological, immunological
and microbiological examinations. Moreover, the traditional culture and serological
methods for detection of abortion agents are not highly sensitive and rapid. Therefore,
the molecular methods such as real-time PCR have been used for detection of bovine
abortion infectious agents. [77, 146].
In this study, a TaqMan real-time PCR-based panel for detection of the most
important infectious abortion agents in cattle named as Dembo abortion-PCR was
developed. This system can detect all target abortogenic agents in a single run with
rapidity, high sensitivity, and specificity.
21
I.2. Materials and methods
I.2.1. Primers and probes design
Twenty-four bovine abortogenic pathogens including 11 viruses, 8 bacteria, 4
protozoa, and 1 fungus including BVDV, IBAV, Simbu group viruses (Schmallenberg
virus, Douglas virus, Shamonda virus and Sathuperi virus) AKAV, CHUV, BHV-1,
BTV, Aino virus, S. enterica ser. Dublin, S. enterica ser. Typhimurium, S. enterica ser.
Enteritidis, C. abortus, B. abortus, C. fetus subsp. venerealis, L. monocytogenes,
Leptospira spp., T. gondii, T. foetus, N. caninum, S. cruzi, and Aspergillus spp. were
selected as target pathogens of the assay.
In this study, primers and probes were designed for 9 out of 24 target pathogens.
The newly designed assays were for following pathogens: Simbu group viruses
(Schmallenberg virus, CHUV, Sathuperi virus, Shamonda virus), IBAV, Douglas virus,
Aino virus, T. gondii and N. caninum. One same set of primers and probes were
designed to detect the Simbu group viruses including Schmallenberg virus, Sathuperi
virus, Douglas virus and Shamonda virus. The conserved region in the genome of each
pathogen was downloaded from NCBI database, and new sets of primer and probes were
designed using PrimerQuest software (Integrated DNA Technologies, Inc., Coralville,
IA, U.S.A.) based on consensus sequence which was acquired by multiple alignments
using BioEdit tool. The primers and probes for 15 other pathogens were selected from
previously developed assays which were associated with qPCR [58, 98, 109, 117, 122,
130, 137, 139, 146, 154, 159]. A previously reported set of primers and probe for β-
actin was used as perfect nucleic acid extraction indicator [86, 146, 155]. All used
probes were indicated by dye FAM (6-carboxyfluorescein) at the 5′ end and the
fluorescent dye TAMRA (6-carboxytetramethylrhodamine) at the 3′ end. Primers and
probes were manufactured at Sigma-Aldrich (St. Louis, MO, U.S.A.), and probes with
mixed bases were ordered from Integrated DNA Technologies. Information about all
primers and probes used in this study is listed in Table 1.1
22
I.2.2. Preparation of genomic DNA and RNA
DNA and RNA extraction was done using two commercial kits, according to the
manufacturer’s instructions. Extraction of viral DNA and RNA was performed using
High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany),
and the bacterial, protozoal and fungal DNAs were extracted by DNeasy blood and
tissue Kit (Qiagen, Hilden, Germany), The extracted DNA and RNA were stored at
−80°C unit testing by Dembo-PCR.
I.2.3. Real-time PCR amplification
A One Step PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa Bio, Otsu,
Japan) was used to amplify viral RNA, and Premix Ex Taq (Perfect Real Time)
(TaKaRa Bio) was used for amplification of the viral, protozoal, fungal and bacterial
DNA. Reactions were performed in a total volume of 20 μl of sample nucleic acid,
primers, probes (the final concentration of all primers and probes was 0.2 μM) and other
components including the kits, according to the manufacturers’ instructions. Condition
of thermal cycling were as follows: 45°C for 5 min and 95°C for 30 sec, followed by 40
cycles of 95°C for 10 sec, 55°C for 20 sec and 72°C for 20 sec. The automatic analytic
option was used in LightCycler Nano Software 1.1 (Roche Diagnostics GmbH) to
analyze the fluorescence data. The parameters of analysis were as follows: Exclude
early cycle = 7, minimum relative amplifications = 0, and minimum amplification
quality = 5.
I.2.4. Sensitivity test
To evaluate the real-time PCR performance, the tenfold serial dilution of
synthesized DNA of all target pathogens, containing target genome regions (1×100–1×
106 or 5 × 100–5 × 106 copies/reaction) were used to determine the limit of detection
(LOD), correlation coefficient (R2), and PCR efficiency (E) as important parameters of
TaqMan real-time PCR. The synthesized DNA was fabricated at Integrated DNA
23
Technologies. Pathogen dilutions were repeated twice in separate runs, and a standard
curve was constructed from the Cq values.
PCR efficiency (E) was calculated using the standard curve slope according to the
following formula: E= (10-1/slope (-1)). The correlation coefficient (R2) was also
calculated.
I.2.5. Specificity test
To avoid false positive results and to guarantee that the assays only react with
the specific genome regions of target pathogens, the specificity of all sets of primers
and probes were checked. A total of 21 isolated strains of bovine pathogens were tested
for individual sets of primers and probes of target pathogens (Table 1.3).
I.2.6. Clinical samples
I.2.6.1. Blood samples
A total of 22 blood samples from 19 clinically healthy cattle and 3 aborted cattle
were collected from 2 cattle farms on August 2016 in Ibaraki prefecture, Japan. The
nucleic acids were extracted from each sample and analyzed by Dembo abortion-PCR.
I.2.6.2. Aborted fetus samples
A total of 40 bovine aborted fetus samples and 2 swine aborted fetus samples,
including spinal cord, brainstem, cerebrum, liver, lung, kidney and spleen were
collected from Kagoshima and Miyazaki areas in Japan. DNAs and RNAs of all samples
were extracted by the commercial extraction kits as described above, then analyzed by
Dembo abortion-PCR.
I.3. Results
I.3.1. Sensitivity of the Dembo abortion-PCR
Table 1.2, and Fig. 1.1 and 1.2 (A, B and C), show the LOD, R2, and E value of
the Dembo abortion-PCR using the LightCycler Nano instrument. The LOD, according
24
to the DNA copy number of all pathogens, was between 1–100 copies per reaction. The
coverage of calibration curves for each assay was within a linear dynamic range of more
than five orders of magnitude, and R2 values were at least [0.9582]. E values were in the
range of 92.1–106%.
I.3.2. Specificity of Dembo abortion-PCR
All sets of primers and probes were able to detect only the targeted region of the
particular pathogens, which means, all assays were highly specific (Fig.1.3 A, B, and
C).
I.3.3. Clinical samples
I.3.3.1. Blood samples
All 22 blood samples were negative by Dembo abortion-PCR (Tables 1.4.A and
B).
I.3.3.2. Aborted fetus samples
Only AKAV was detected in 2 swine aborted fetus samples from Miyazaki
prefecture by Dembo abortion-PCR (Fig. 1.4). On the other hand, none of the pathogens
were detected in 40 bovine aborted fetus samples by Dembo abortion-PCR (Table 1.5).
I.4. Discussion
In the study, I developed a new system for simultaneous detection of multiple
agents causing abortion in cattle. This system was nominated as a detection system for
microbes from bovine abortion by real-time PCR (referred to as Dembo abortion-PCR).
Dembo- abortion PCR is able to detect a total of 24 cattle abortogenic agents in a single
run, including 11 viruses, 8 bacteria, 4 protozoa, and 1 fungus, within 3 hours. Primers
and probes were newly designed for 9 out of 24 target pathogens. The newly designed
25
primers and probes were for following pathogens: CHUV, Sathuperi virus, Shamonda
virus, IBAV, Douglas virus, Aino virus, Toxoplasma gondii and Neospora caninum.
A total of 22 blood samples were collected from 2 cattle farms on August 2016
in Ibaraki prefecture, Japan and screened by Dembo abortion-PCR. All 22 blood
samples were negative for all Dembo abortion-PCR targeted pathogens. Since detection
of abortogenic agents from blood samples by PCR is not reported so far, it is likely that
blood samples are not suitable samples for detection of abortogenic pathogens by
Dembo abortion-PCR. Subsequently, 40 bovine aborted fetus samples and 2 pig aborted
fetus samples, including spinal cord, brainstem, cerebrum, liver, lung, kidney and spleen
were screened. All 40 bovine aborted fetus samples were negative for any pathogens by
Dembo abortion-PCR.
Ideally, for collecting samples, the intact aborted fetus, placenta and serum
samples from the dam are the optimal specimens, thus; the whole fetus and placenta
should be saved and placed in a clean bag, which should then be refrigerated as soon as
possible [9, 13]. The inclusion of the placenta is critical in the diagnosis of some mycotic
and bacterial abortions where the placenta is the primary tissue affected [9]. First
sampling should be done as soon as possible after the abortion is noted, with the second
sample being collected in 2-4 weeks [9, 13, 29]. In spite of these facts, bovine abortion
has infectious and non-infectious causes [110, 124], thus; it may have indicated that the
sampling methods in this study were not accurate enough or the causes of abortions
were non- infectious. However; AKAV was detected in the 2 pig aborted fetus samples.
AKAV is an arthropod-borne virus, which belongs to the genus Orthobunyavirus in the
family Peribunyaviridae [75]. AKAV in cattle, sheep, goats, and pig is responsible for
stillbirths, abortions, congenital arthrogryposis-hydranencephaly syndrome, and
hydranencephaly micrencephaly syndrome [75]. Thus, AKAV was the causative agent
of abortion of the 2 pig fetus.
26
In summary, Dembo abortion-PCR was developed for identification and
detection of a wide range of existing pathogens. The sensitivity, and specificity of all
assays were high and ideal using synthesized DNAs and 21 isolated pathogens from
cattle, respectively. However, all clinical samples including 22 blood samples from
cattle and 40 aborted fetus samples from cattle were negative by Dembo abortion-PCR,
where only 2 aborted fetus samples from pigs were positive for Akabane virus. Thus,
farther studies are needed for validation of Dembo abortion-PCR using clinical samples.
27
Tab
le 1
.1. I
nfor
mat
ion
on a
ll pr
imer
s and
pro
bes u
sed
in c
urre
nt st
udy
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
/TA
MR
A)
Ref
eren
ce N
o.
Ain
o vi
rus
M p
olyp
rote
in
Forw
ard
AG
CA
AA
TCC
CA
TTG
CG
TGA
Th
is st
udy
Rev
erse
C
AG
AC
TTC
TGC
TGG
CA
CA
TTA
Prob
e A
GG
GA
CA
AC
TGG
CTC
TCG
CT
Aka
bane
viru
s S
segm
ent
Forw
ard
TCA
AC
CA
GA
AG
AA
GG
CC
AA
GA
T 13
7
Rev
erse
G
GG
AA
AA
TGG
TTA
TTA
AC
CA
CTG
TAA
A
Prob
e TT
AC
ATA
AG
AC
GC
CA
CA
AC
CA
Blu
eton
gue
viru
s N
S3 g
ene
Forw
ard
AA
ATM
TTG
GA
YA
AA
GC
RA
TGTC
AA
A
153
Rev
erse
C
TYA
CR
TCA
TCA
CG
AA
AC
GC
T
Prob
e A
AR
GC
TGC
ATT
CG
CA
TCG
TAC
GC
Chu
zan
viru
s V
P7 g
ene
Forw
ard
TGA
TCG
AA
CG
CC
AA
CA
CTT
Th
is st
udy
Rev
erse
G
GC
AA
TCC
AA
CC
CTC
ATA
CA
Prob
e TA
TCA
CC
AC
AA
TGG
CA
TGC
ATT
GC
G
Ibar
aki v
irus
VP3
gen
e Fo
rwar
d TA
CA
GC
GG
GA
CC
TAG
GTT
TA
This
stud
y
Rev
erse
G
TTC
TCC
CG
TTG
GA
CC
ATA
TT
Prob
e TG
GC
AC
GA
CA
GC
TTG
ATA
TTG
CC
T
Sim
bu g
roup
* N
, NSs
gen
e, S
segm
ent
Forw
ard
TGA
AG
ATG
TAC
CA
CA
AC
GG
AA
T Th
is st
udy
Rev
erse
G
AG
GA
AG
AA
GA
CTC
TAG
CA
AC
AC
Prob
e A
CC
TCC
GG
GTT
AA
ATG
TAG
CTG
C
Aspe
rgill
us sp
p.
18S
ribos
omal
RN
A g
ene
Forw
ard
GC
CC
GC
CG
TTTC
GA
C
98
Rev
erse
C
CG
TTG
TTG
AA
AG
TTTT
AA
CTG
ATT
AC
Prob
e C
CCG
CC
GA
AG
AC
CC
CA
AC
ATG
Bruc
ella
abo
rtus
omp2
a ge
ne
Forw
ard
GC
GG
CTT
TTC
TATC
AC
GG
TATT
C
122
Rev
erse
C
ATG
CG
CTA
TGA
TCTG
GTT
AC
G
Prob
e C
GC
TCA
TGC
TCG
CC
AG
AC
TTC
AA
TG
Cam
pylo
bact
er fe
tus s
ubsp
. ven
erea
lis
nahE
gen
e Fo
rwar
d TT
CA
AA
AG
CTC
TTG
GG
GTT
AC
58
Rev
erse
A
AA
GC
CTT
GTT
TAG
AA
CA
ATA
TAA
CTC
Prob
e A
CTC
GTG
GTG
GA
GA
GC
GTA
G
28
Tab
le 1
.1. C
ontin
ued
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
/TA
MR
A)
Ref
eren
ce N
o.
Chl
amyd
ophi
la a
bortu
s om
pA g
ene
Forw
ard
GC
AA
CTG
AC
AC
TAA
GTC
GG
CTA
CA
11
7
Rev
erse
A
CA
AG
CA
TGTT
CA
ATC
GA
TAA
GA
GA
Prob
e TA
AA
TAC
CA
CG
AA
TGG
CA
AG
TTG
GTT
TAG
CG
Lept
ospi
ra sp
p.
lipL3
2 ge
ne
Forw
ard
AA
GC
ATT
AC
CG
CTT
GTG
GTG
13
9
Rev
erse
G
AA
CTC
CC
ATT
TCA
GC
GA
T
Prob
e A
AA
GC
CA
GG
AC
AA
GC
GC
CG
List
eria
mon
ocyt
ogen
es
iap
gene
Fo
rwar
d C
ATG
GC
AC
CA
CC
AG
CA
TCT
130
Rev
erse
A
TCC
GC
GTG
TTTC
TTTT
CG
A
Prob
e C
GC
CTG
CA
AG
TCC
TAA
GA
CG
CC
A
Neo
spor
a ca
ninu
m
NC
5 ge
ne
Forw
ard
GG
GA
TAC
GTG
GTT
TGTG
GTT
AG
Th
is st
udy
Rev
erse
C
AC
AG
AA
CA
CTG
AA
CTC
TCG
ATA
AG
Prob
e TC
AC
GTT
GA
AA
TCA
GC
CTG
CG
TCA
Sarc
ocys
tis c
ruzi
18 s
ribos
omal
RN
A g
ene
Forw
ard
TCTG
CTG
GA
AG
CA
ATC
AG
TC
109
Rev
erse
TT
GA
AG
CA
GG
CTT
ATT
GC
CT
Prob
e A
CC
CA
TCTA
TATT
GG
GA
TAA
TAC
CG
TTA
CT
Toxo
plas
ma
gond
ii p3
0 ge
ne
Forw
ard
GC
CTC
ATC
GG
TCG
TCA
ATA
A
This
stud
y
Rev
erse
G
TCA
TTG
TAG
TGG
GTC
CTT
CC
Prob
e A
GC
AC
TCTT
GG
TCC
TGTC
AA
GTT
GT
Tritr
icho
mon
as fo
etus
5.
8S ri
boso
mal
RN
A G
ENE
Forw
ard
GC
GG
CTG
GA
TTA
GC
TTTC
TTT
158
Rev
erse
A
TGC
AC
ATT
GC
GC
GC
C
Prob
e A
CA
AG
TTC
GA
TCTT
TG
Salm
onel
la D
ublin
V
agC
Fo
rwar
d G
GG
TGA
GC
GA
GC
TGG
AA
A
146
Rev
erse
C
GC
CA
TAA
AG
TCC
GG
GTC
A
Prob
e TT
TTTC
GA
GC
TGC
GC
GA
AC
GA
GC
Salm
onel
la T
yphi
mur
ium
Fi
c Fo
rwar
d TG
CA
GA
AA
ATT
GA
TGC
TGC
T 14
6
Rev
erse
TT
GC
CC
AG
GTT
GG
TAA
TAG
C
Prob
e A
CC
TGG
GTG
CG
GTA
CA
GA
AC
CG
T
29
T
able
1.1
. Con
tinue
d
*Sim
bu g
roup
: Dou
glas
viru
s, Sa
thup
eri v
irus,
Sham
onda
viru
s, Sc
hmal
lenb
erg
viru
s.
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
/TA
MR
A)
Ref
eren
ce N
o.
Salm
onel
la E
nter
itidi
s Se
fA
Forw
ard
GG
TAA
AG
GG
GC
TTC
GG
TATC
14
6
Rev
erse
TA
TTG
GC
TCC
CTG
AA
TAC
GC
Prob
e TG
GTG
GTG
TAG
CC
AC
TGTC
CC
GT
Bov
ine
herp
esvi
rus 1
gE
Fo
rwar
d C
AA
TAA
CA
GC
GTA
GA
CC
TGG
TC
146
Rev
erse
G
CTG
TAG
TCC
CA
AG
CTT
CC
AC
Prob
e TG
CG
GC
CTC
CG
GG
CTT
TAC
GTC
T
Bov
ine
vira
l dia
rrhe
a vi
rus
5′ U
TR
Forw
ard
GG
GN
AG
TCG
TCA
RTG
GTT
CG
14
6
Rev
erse
G
TGC
CA
TGTA
CA
GC
AG
AG
WTT
TT
Prob
e C
CA
YG
TGG
AC
GA
GG
GC
AY
GC
β-A
CTI
N
Act
in
Forw
ard
AG
CG
CA
AG
TAC
TCC
GTG
TG
155
Rev
erse
C
GG
AC
TCA
TCG
TAC
TCC
TGC
TT
Prob
e TC
GC
TGTC
CA
CC
TTC
CA
GC
AG
ATG
T
30
Table 1.2. Results of sensitivity tests for bovine abortive pathogens obtained by using the LightCycler Nano (Roche Diagnostics)
First reaction Second reaction First reaction
Second reaction First reaction
Second reaction
` Sarcocystis cruzi 1 1 2.026 1.884 0.9827 0.9981
Toxoplasma gondii 10 10 2.048 2.235 0.9984 0.9814
Tritrichomonas fetus 10 10 1.992 1.838 0.9981 0.9738
Neospora caninum 10 100 1.954 1.972 0.9998 0.9679
Campylobacter fetus 10 100 1.909 2.120 0.9991 0.9634
Chlamydophila abortus 1 10 2.031 1.883 0.9582 0.9999
Listeria monocytogenes 10 10 1.941 1.940 0.9994 0.9997
Leptospira spp. 100 10 1.932 1.935 0.9950 0.9996
Brucella abortus 1 10 1.957 2.017 0.9994 0.9998
Bluetongue virus 1 10 2.076 1.987 1 0.9993
Akabane virus 100 100 1.891 1.858 0.9976 0.9993
Aino virus 10 10 1.975 1.878 0.9989 0.9971
Chuzan virus 10 10 1.938 1.944 0.9986 0.9998
Bovine herpes virus-1 100 10 2.159 1.930 0.6052 0.9948
Bovine viral diarrhea virus 100 100 1.930 2.007 0.9927 0.9897
Simbu group* 10 10 1.842 1.863 0.9981 0.9998
Ibaraki virus 10 10 1.921 1.944 0.9993 0.9980
Aspergillus spp. 1 1 2.130 2.080 0.9170 0.9900
Salmonella Enteritidis 10 10 1.962 2.159 0.9980 0.9550
Salmonella Typhimurium 100 100 2.192 2.173 0.9950 0.9960
Salmonella Dublin 10 10 1.958 1.930 0.9850 0.9910
*Simbu group: Douglas virus, Sathuperi virus, Shamonda virus, Schmallenberg virus.
Efficiencycciencyy Coefficient of DeterminationationLimit of detection (copies/reaction)
itectionaction))
Target pathogenhogen
31
Table 1.3. Isolated bovine pathogens, used for specificity tests.
No. Isolated pathogen
1 Akabane virus
2 Aino virus
3 Bovine enterovirus u31
4 Bovine enterovirus p5
5 Coronavirus
6 Torovirus
7 Adenovirus
8 Ibaraki virus
9 Reo virus
10 Bovine viral diarrhea virus
11 Bovine herpes viurs-1
12 Clostridium perfringens
13 Mycobacterium avium spp. paratuberculosis
14 Salmonella enteritidis
15 Neospora caninum
16 Listeria monocytogenes
17 Salmonella Dublin
18 Salmonella Typhimurium
19 Toxoplasma gondii
20 Sarcocystis cruzi
21 Enterotoxogenic Escherichia coli
32
Table 1.4 (A). Blood samples from first farm, screened in this study
Sample No. Sex Age/Year Abortion Sampling date Detected pathogen 1 Female 10 Yes August 2016 Non 2 Female 2 No August 2016 Non 3 Female 2 No August 2016 Non 4 Female 1 No August 2016 Non 5 Female 4 No August 2016 Non 6 Female 4 No August 2016 Non 7 Female 1 No August 2016 Non 8 Female 1 No August 2016 Non 9 Female 1 No August 2016 Non
10 Female 1 No August 2016 Non 11 Female 1 Yes August 2016 Non
Table 1.4 (B). Blood samples from second farm, screened in this study
Sample No. Sex Age/Year Abortion Sampling date Detected pathogen 1 Female 9 Yes August 2016 Non 2 Female 2 No August 2016 Non 3 Female 13 No August 2016 Non 4 Female 6 Yes August 2016 Non 5 Female 3 No August 2016 Non 6 Female 2 No August 2016 Non 7 Female 5 No August 2016 Non 8 Female 1 No August 2016 Non 9 Female 1 No August 2016 Non
10 Female 1 No August 2016 Non 11 Female 1 No August 2016 Non
33
Sample No. Animal Organ Date of extortion Location Dembo-PCR results
1 Cattle Spinal cord 16.10.24 Kagoshima Negative
2 Cattle Spinal cord 16.10.24 Kagoshima Negative
3 Cattle Spinal cord 16.10.24 Kagoshima Negative
4 Cattle Spinal cord 16.10.24 Kagoshima Negative
5 Cattle Spinal cord 16.10.24 Kagoshima Negative
6 Cattle Spinal cord 16.10.24 Kagoshima Negative
7 Cattle Spinal cord 16.10.24 Kagoshima Negative
8 Cattle Spinal cord 16.10.24 Kagoshima Negative
9 Cattle Spinal cord 16.10.24 Kagoshima Negative
10 Cattle Spinal cord 16.10.24 Kagoshima Negative
11 Cattle Spinal cord 16.10.24 Kagoshima Negative
12 Cattle Spinal cord 16.10.24 Kagoshima Negative
13 Cattle Spinal cord 16.10.24 Kagoshima Negative
14 Cattle Spinal cord 16.10.24 Kagoshima Negative
15 Cattle Spinal cord 16.10.24 Kagoshima Negative
16 Cattle Spinal cord 16.10.24 Kagoshima Negative
17 Cattle Spinal cord 16.10.24 Kagoshima Negative
18 Cattle Cerebrum 16.10.25 Kagoshima Negative
19 Cattle Cerebrum 16.10.25 Kagoshima Negative
20 Cattle Cerebrum 16.10.25 Kagoshima Negative
21 Cattle Cerebrum 16.10.25 Kagoshima Negative
22 Cattle Cerebrum 16.10.25 Kagoshima Negative
23 Cattle Spinal cord 16.10.25 Kagoshima Negative
24 Cattle Cerebrum 16.10.25 Kagoshima Negative
25 Cattle Cerebrum 16.10.25 Kagoshima Negative
26 Cattle Cerebrum 16.10.25 Kagoshima Negative
27 Cattle Cerebrum 16.10.25 Kagoshima Negative
28 Cattle Cerebrum 16.10.25 Kagoshima Negative
29 Cattle Cerebrum 16.10.25 Kagoshima Negative
30 Cattle
Cerebrum 16.10.25 Kagoshima Negative
Table 1.5. Results of aborted fetus samples
34
Sample No. Animal Organ Date of extortion Location Dembo-PCR results
31 Cattle Brainstem 16.10.25 Kagoshima Negative
32 Cattle Cerebrum 16.10.25 Kagoshima Negative
33 Cattle Cerebrum 16.10.25 Kagoshima Negative
34 Cattle Cerebrum 16.10.25 Kagoshima Negative
35 Cattle Cerebrum 16.10.25 Kagoshima Negative
36 Cattle Cerebrum 16.10.25 Kagoshima Negative
37 Cattle Liver 16.10.25 Kagoshima Negative
38 Cattle Lung 16.10.25 Kagoshima Negative
39 Cattle Kidney 16.10.25 Kagoshima Negative
40 Cattle Spleen 16.10.25 Kagoshima Negative
1 Pig Brainstem 16.10.24 Miyazaki Akabane virus
2 Pig Brainstem 16.10.24 Miyazaki Akabane virus
Table 1.5. Continued
35
105 10
4 10
3 10
2
101
Fig. 1.1. Limit of detection using Nano instrument
106
36
Fig. 1.2 (A). Standard curves using synthesized DNA
Cq
valu
e
Cq v
alue
Log copy number per reaction
Akabane virus
Log copy number per reaction
Aino virus
BVDV
Log copy number per reaction
Cq
valu
e
Chuzan virus
Cq v
alue
Log copy number per reaction
Ibarake virus
Cq
valu
e
Log copy number per reaction
Simbu Group viruses
Log copy number per reaction
Cq
valu
e
BHV-1
Log copy number per reaction
Cq
valu
e
Bluetongue virus
Log copy number per reaction
Cq
valu
e
37
Fig. 1.2 (B). Standard curves using synthesized DNA
Cq
valu
e
Log copy number per reaction
Brucella abortus Chlamydophila abortus
Cq
valu
e
Log copy number per reaction
Champylobacter fetus
Cq
valu
e
Log copy number per reaction
Leptospira spp.
Log copy number per reaction
Cq
valu
e
Listeria monocytogenes
Cq
valu
e
Log copy number per reaction
Salmonella enetritidis
Log copy number per reaction
Cq
valu
e
Salmonella Typhimurium
Cq
valu
e
Log copy number per reaction
Salmonella Dublin
Log copy number per reaction
Cq
valu
e
38
Fig. 1.2 (C). Standard curves using synthesized DNA
Fig. 1.2 (A), (B) and (C). Standard curves obtained using synthesized DNA. Cq values were plotted against the log copy number of synthesized DNA. The regression curve (y), correlation coefficient (R2) and E value (E) were calculated.
Sarcocystis cruzi
Log copy number per reaction
Cq
valu
e
Toxoplasma gondii
Cq
valu
e
Log copy number per reaction
Neospora caninum
Cq
valu
e
Log copy number per reaction
Tritrichomonas fetus
Cq
valu
e
Log copy number per reaction
Aspergillus spp.
Cq
valu
e
Log copy number per reaction
39
Fig. 1.3 (A). Specificity tests results
Aino Virus Akabane Virus
BVDV Chuzan virus
Ibaraki virus BHV-1
Simbu group viruses Bluetongue virus
Champylobacter fetus Brucella abortus
40
Fig. 1.3 (B). Specificity tests results
Chlamydophila abortus Leptospira Spp.
Listeria monocytogenes Salmonella Dublin
Salmonella enteritidis Salmonella Typhimurium
Sarcocystis cruzi Toxoplasma gondii
Tritrichomonas fetus Neospora caninum
41
Fig. 1.3 (C). Specificity tests results
Fig. 1.2 (A), (B) and (C). Specificity test results of all targeted abortogenic pathogens using 21 isolated bovine pathogens.
Aspergillus Spp.
42
.
Fig. 1.4(A). Akabane virus detected from pig aborted fetus-
sample by Dembo abortion-PCR
1; beta-actin (DNA), Cq = 19.082. 2; beta-actin (RNA), Cq = 31.286.
3; Akabane virus, Cq = 33.544
Fig. 1.4(B). Akabane virus detected from pig aborted fetus-
sample by Dembo abortion-PCR
1; beta-actin (DNA), Cq = 17.153. 2; beta-actin (RNA), Cq = 31.972.
3; Akabane virus, Cq = 32.297
1
2
3
1
2
3
43
CHAPTER TWO
« Detection of Infectious Agents of Abortion, and Respiratory Disease Complex
in Potential Vectors and Reservoirs with Dembo-PCR »
44
II.1. Introduction
Abortion, and respiratory infectious agents cause a broad spectrum of diseases,
resulting in significant economic losses in the cattle industry [54, 69, 107]. In the US, it
has been estimated that late-term cattle abortion costs between US $500–900 per case
and that the cattle respiratory disease complex causes 70–80% of all feedlot cattle
morbidity and 40–50% of all cattle mortality, resulting in major economic losses
amounting to more than $500 million per year [71, 107].
Various vectors and reservoirs play important roles in the transmission of many
pathogens [33, 114]. Vector-borne diseases could be transmitted by insects such as
mosquitoes, flies, ticks, fleas, and lice [33, 114]. Vectors are divided into two types:
biological and mechanical vectors. Biological vectors carry infectious agents or
pathogens within their bodies, where the infectious agents undergo multiplication and/or
development, consequently transmitting the infectious agents to the host through bites.
Mosquitoes represent a biological vector of many pathogens. Mechanical vectors
transfer pathogens from an infected host or a contaminated substrate to a susceptible
host without multiplying and/or developing of the pathogens within the vector [124].
Many insects can serve as mechanical vectors [14, 40, 88]. Apart from vectors described
above, many infectious diseases of cattle are considered to be maintained in other
reservoir hosts including other mammals such as rodents, ruminants and carnivores [68]
Several studies showed that vectors and reservoirs play a critical role in the
transmission of a broad spectrum of pathogens, including BVDV, BEV, AKAV,
Salmonella enterica ser. Typhimurium, E. coli, and Campylobacter spp [23, 40, 56, 88,
114].
In the Chapter one, detection system of microbes for bovine abortion diseases
by real-time PCR (Dembo abortion-PCR) was developed. Addition to this system,
Dembo diarrhea-PCR and Dembo respiratory-PCR system were developed previously
[86, 146]. These Dembo-PCR systems exhibit high sensitivity, high specificity, rapidity,
45
and ability to detect all targeted infectious agents simultaneously.
In this Chapter, I evaluated by Dembo-PCR, especially in Dembo respiratory-
and abortion-PCR, whether the infectious agents, causing diseases in cattle, could be
transmitted by vectors and reservoirs such as insects, arthropods, rodents and birds and
particularly targeted a total of 31 pathogens including IBAV, Simbu group viruses
(Schmallenberg virus, Douglas virus, Shamonda virus, Sathuperi virus), AKAV,
CHUV, BTV, Aino virus, BPIV 3, BAdV 3, BRSV, IDV, BRAV, BRBV, M.
haemolytica, H. somni, Trueperella pyogenes, C. abortus, B. abortus, C. fetus subsp.
venerealis , L. monocytogenes, M. bovis, U. diversum, Pasteurella multocida,
Leptospira spp., T. gondii, T. foetus, N. caninum, S. cruzi, and Aspergillus spp.
II.2. Materials and methods
II.2.1. Primer and probe design
I selected 31 pathogens as bovine abortogenic, and respiratory disease complex
infectious agents. To detect 22 pathogens, previously reported primers and probes were
used [58, 86, 98, 109, 117, 122, 130, 137, 139, 146, 154, 159]. We used newly designed
primers and probes for the remaining 9 pathogens, including the Simbu group viruses
(Schmallenberg virus, Sathuperi virus, Shamonda virus, Douglas virus), CHUV, IBAV,
Aino virus, Toxoplasma gondii and Neospora caninum as mentioned in Chapter two.
All probes were indicated by the dye FAM (6-carboxyfluorecein) at the 5´ end and the
fluorescent dye TAMRA (6-carboxytetramethylrhodamine) at the 3´ end. All primers
and probes were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and Integrated
DNA Technologies. The primers and probes used in the study were listed in Table 2.1.
II.2.2. Extraction of nucleic acids
Viral nucleic acids were extracted from samples using High Pure Viral Nucleic
Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany). QIAamp Fast DNA Stool
Mini Kit (Qiagen, Hilden, Germany) was used to extract bacterial, protozoal, and fungal
46
DNA from specimens, according to the manufacturer’s instructions. Extracted DNA and
RNA were stored at −80°C until use.
II.2.3. Real-time PCR amplification
All TaqMan real-time PCR assays were performed under the same reaction
condition used for the Dembo abortion-PCR, as described in Chapter one [86, 146]. A
One Step PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa Bio, Otsu, Japan) was
used to detect viral RNA, and Premix Ex Taq (Perfect Real Time) (TaKaRa Bio) was
used to detect the viral, protozoal, fungal, and bacterial DNAs. The real-time PCR assay
was performed with the Applied Biosystems 7300 Real-Time PCR System (ABI 7300)
for screening and with the LightCycler Nano (Roche Diagnostics GmbH) for validation
of positive samples during screening. To analyze the fluorescence data, automatic
analysis option was used in the LightCycler Nano Software 1.1 (Roche Diagnostics
GmbH) and the Applied Biosystems 7300 Real-Time PCR software, respectively.
II.2.4. Analysis of field samples
A total of 117 vector and reservoir samples, including 64 flies, 18 gadflies, 7
arthropods, 14 fecal and intestinal contents from rodents, and 14 fecal samples of birds
were collected from inside and outside of 4 dairy cattle farms and 18 beef cattle farms
between 2014 to 2016 in Japan (Table 2.2). The nucleic acids were extracted from each
sample. To detect bovine abortive and respiratory disease complex pathogens, extracted
nucleic acid samples were pooled as shown in Table 2.3. RNAs in each pooled sample
were reverse transcribed into complementary DNA (cDNA) by using SuperScript™ III
Reverse Transcriptase (Invitrogen), and then cDNA and genomic DNA were amplified
by using Genomiphi V2 DNA Amplification Kit (GE Healthcare). The extracted nucleic
acids were evaluated in triplicates with Dembo-PCR [144]. When the Cq values were
calculated with the built-in algorithm in two or more runs out of three, the samples were
considered to be positive.
47
II.3. Results
II.3.1. Sensitivity and specificity of the Dembo abortion-PCR
LOD, R2, and E value of the Dembo abortion-PCR using the LightCycler Nano
instrument was summarized in the Chapter one (Table 1.3). In the ABI 7300 instrument,
the LOD of the Dembo respiratory- and abortion-PCR was evaluated with 100 copies
per reaction. When the sensitivity was lower than 100 copies per reaction, lower diluents
were used to evaluate LOD. Out of 36 sets of primers and probes, 31 sets showed the
sensitivity of 100 copies per reaction, while other 5 sets showed the sensitivity of 250
copies per reaction (Table 2.4).
II.3.2. Analysis of field samples by Dembo-PCR
To detect abortive and respiratory disease complex pathogens, 15 pooled
samples including 10 pooled samples from flies, 2 pooled samples from gadflies, 2
pooled samples from feces of rodents, 2 pooled samples from feces of birds, and 1
pooled sample from arthropods were screened in the ABI 7300 instrument. N. caninum
was detected only in an arthropod pooled sample, which consisted of 7 different
arthropods samples including 2 cockroaches, 2 spiders, and 3 unidentified arthropods,
while all the other pathogens were negative in all the pooled samples. To determine
which arthropods were positive for N. caninum, all 7 arthropod samples were analyzed
in the LightCycler Nano instrument, and found that, the cockroach sample showed
positive reaction exclusively. Table 2.5 shows the results obtained from pooled samples
by Dembo-PCR.
II.4. Discussion
This is the first study that simultaneously evaluates the presence of a wide-range
of bovine pathogens in potential vectors and reservoirs by using Dembo abortion-PCR,
a highly sensitive and rapid pathogen detection system. Dembo abortion-PCR was
performed under the same reaction condition as Dembo diarrhea-PCR and the Dembo
48
respiratory-PCR [86, 146]. I first used Dembo abortion-PCR to detect 24 cattle abortive
agents, including 11 viruses, 8 bacteria, 4 protozoa, and 1 fungus. Subsequently, 50
bovine abortive, diarrheal, and respiratory disease complex pathogens, including 26
viruses, 17 bacteria, 6 protozoa, and 1 fungus, were targeted in a single run by Dembo-
PCR. In Dembo abortion-PCR, the same set of primers and probes were designed to
detect the Schmallenberg virus, Sathuperi virus, Douglas virus and Shamonda virus
[157]. These viruses belong to the Simbu group, which includes important viruses
causing abortion in cattle [158].
From pooled samples, only N. caninum was detected in arthropod pooled
sample, where all other pooled samples were negative using Dembo abortion-PCR and
Dembo-respiratory-PCR (Table 2.5). To determine which arthropods were positive for
N. caninum, all 7 arthropod samples were analyzed in the LightCycler Nano instrument,
and found that, the cockroach sample showed positive reaction exclusively.
N. caninum results were confirmed by Nested PCR (fig. 2.1), and sequences
were obtained by cycle sequencing (data not shown). N. caninum is an obligate
intracellular coccidian parasite that is globally distributed and is one of the major
pathogens that cause abortion in cattle [94, 143]. A broad spectrum of wild and domestic
animals can be infected by N. caninum. Dogs, coyotes, and gray wolves (Canis lupus)
are considered to be the final hosts of N. caninum. However, mammals and birds,
including cattle, sheep, goat, water buffalo, horse, donkey, bison, white-tailed deer, red
fox, chicken, pigeon, sparrow, feral swine, capybara, and rabbit can serve as potential
natural intermediate hosts for this pathogen. Although N. caninum has been detected in
several mammals and birds, further investigation regarding the lifecycle and hosts of
this pathogen is required [48, 94, 134, 143]. Cockroaches are the vectors or potential
transmitters of protozoans such as T. gondii, Sarcocystis oocysts, and others [87].
In this study, I detected N. caninum genome in a cockroach sample for the first
time, implying that cockroaches may play a role in its life cycle, so that they may serve
49
as a potential vector of N. caninum.
To detect 19 bovine diarrheal pathogens, all 117 samples were individually
screened using Dembo abortion-PCR by Dr. Shinobu Tsuchiaka, who belonged to
Research and Education Center for Prevention of Global Infectious Diseases of
Animals, Tokyo University of Agriculture and Technology. He detected genome of
BVDV, BEV, Salmonella enterica ser. Dublin, and Salmonella enterica ser.
Typhimurium from various vectors and reservoirs including flies, rodent, arthropods,
and birds [145].
In his findings, BVDV was one of the most frequently detected pathogens from
a wide spectrum of vectors and reservoirs, including flies, gadflies, and rodent and bird
fecal matter. It should be noted that in addition to diarrhea, BVDV causes abortion and
respiratory diseases [86, 97, 146]. Out of 117 vector and reservoir samples, twenty
samples were tested positive for BVDV, and flies formed the largest group among the
vectors and reservoirs with 8 positive cases of BVDV. The data is consistent with the
previous study that reported flies as a potential source of BVDV transmission in cattle
[23]. BVDV was also detected in rodent and avian fecal matter, whereas no previous
study reported the presence of BVDV in rodents and birds. All the studies (current and
past [86, 146]) may imply that BVDV is one of the most important infectious agents in
cattle-related diseases and that they could be transmitted by flies, rodents, and birds.
Among his analyzed samples, BEV was positive in 34 samples, including 28
flies, 2 gadflies, 1 rodent fecal matter sample, 2 avian fecal matter samples, and 1
cockroach. BEV, which is one of the common viruses in the environment, is very stable
under a broad range of ecological conditions such as changes in pH, temperature, and
salinity. These physiological properties of BEV facilitate easy transmission of BEV to
cattle [93]. Although BEV was detected as diarrheal infectious agent in the study, this
virus also has a potential to cause abortion in cattle [124, 146]. While previous studies
mentioned that BEV could spread in the environment and contaminate water and food,
50
the present study represents the first detection of BEV in vectors and reservoirs [82].
He also detected S. enterica ser. Dublin and S. enterica ser. Typhimurium in
flies, gadflies, and fecal samples of rodents, and birds, suggesting that they may serve
as reservoirs [4, 113, 114]. These serovars of Salmonella causes diarrhea and potentially
also causes abortion and respiratory diseases in cattle [13, 86, 146].
In this study, I did not demonstrate whether cattle in the farms were infected
with these pathogens. Further study is needed to isolate these bacteria by culture from
potential vectors and reservoirs because only bacterial genomes were detected in this
study, and to investigate the transmission from these hygiene pests to cattle.
The data showed that arthropods and rodents, while acting as potential vectors
and reservoirs of cattle pathogens, can carry more than one pathogen at the same time.
This is the first demonstration of vectors and reservoirs acting in tandem to transmit
several infectious agents
51
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Ain
o vi
rus
M p
olyp
rote
in
Forw
ard
AG
CA
AA
TCC
CA
TTG
CG
TGA
Th
is st
udy
Rev
erse
C
AG
AC
TTC
TGC
TGG
CA
CA
TTA
Prob
e A
GG
GA
CA
AC
TGG
CTC
TCG
CT
Aka
bane
viru
s S
segm
ent
Forw
ard
TCA
AC
CA
GA
AG
AA
GG
CC
AA
GA
T 13
7
Rev
erse
G
GG
AA
AA
TGG
TTA
TTA
AC
CA
CTG
TAA
A
Prob
e TT
AC
ATA
AG
AC
GC
CA
CA
AC
CA
Blu
eton
gue
viru
s N
S3 g
ene
Forw
ard
AA
ATM
TTG
GA
YA
AA
GC
RA
TGTC
AA
A
154
Rev
erse
C
TYA
CR
TCA
TCA
CG
AA
AC
GC
T
Prob
e A
AR
GC
TGC
ATT
CG
CA
TCG
TAC
GC
Chu
zan
viru
s V
P7 g
ene
Forw
ard
TGA
TCG
AA
CG
CC
AA
CA
CTT
Th
is st
udy
Rev
erse
G
GC
AA
TCC
AA
CC
CTC
ATA
CA
Prob
e TA
TCA
CC
AC
AA
TGG
CA
TGC
ATT
GC
G
Ibar
aki v
irus
VP3
gen
e Fo
rwar
d TA
CA
GC
GG
GA
CC
TAG
GTT
TA
This
stud
y
Rev
erse
G
TTC
TCC
CG
TTG
GA
CC
ATA
TT
Prob
e TG
GC
AC
GA
CA
GC
TTG
ATA
TTG
CC
T
Sim
bu g
roup
* N
, NSs
gen
e, S
segm
ent
Forw
ard
TGA
AG
ATG
TAC
CA
CA
AC
GG
AA
T Th
is st
udy
Rev
erse
G
AG
GA
AG
AA
GA
CTC
TAG
CA
AC
AC
Prob
e A
CC
TCC
GG
GTT
AA
ATG
TAG
CTG
C
Aspe
rgill
us sp
p.
18S
ribos
omal
RN
A g
ene
Forw
ard
GC
CC
GC
CG
TTTC
GA
C
98
Rev
erse
C
CG
TTG
TTG
AA
AG
TTTT
AA
CTG
ATT
AC
Prob
e C
CCG
CC
GA
AG
AC
CC
CA
AC
ATG
Bruc
ella
abo
rtus
omp2
a ge
ne
Forw
ard
GC
GG
CTT
TTC
TATC
AC
GG
TATT
C
122
Rev
erse
C
ATG
CG
CTA
TGA
TCTG
GTT
AC
G
Prob
e C
GC
TCA
TGC
TCG
CC
AG
AC
TTC
AA
TG
Cam
pylo
bact
er fe
tus s
ubsp
. Ven
erea
lis
nahE
gen
e Fo
rwar
d TT
CA
AA
AG
CTC
TTG
GG
GTT
AC
58
Rev
erse
A
AA
GC
CTT
GTT
TAG
AA
CA
ATA
TAA
CTC
Prob
e A
CTC
GTG
GTG
GA
GA
GC
GTA
G
Tab
le 2
.1. I
nfor
mat
ion
on a
ll pr
imer
s and
pro
bes u
sed
in c
urre
nt st
udy
52
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Chl
amyd
ophi
la a
bortu
s
om
pA g
ene
Forw
ard
G
CA
AC
TGA
CA
CTA
AG
TCG
GC
TAC
A
11
7
Rev
erse
A
CA
AG
CA
TGTT
CA
ATC
GA
TAA
GA
GA
Prob
e TA
AA
TAC
CA
CG
AA
TGG
CA
AG
TTG
GTT
TAG
CG
Lept
ospi
ra sp
p.
lipL3
2 ge
ne
Forw
ard
AA
GC
ATT
AC
CG
CTT
GTG
GTG
13
9
Rev
erse
G
AA
CTC
CC
ATT
TCA
GC
GA
T
Prob
e A
AA
GC
CA
GG
AC
AA
GC
GC
CG
List
eria
mon
ocyt
ogen
es
iap
gene
Fo
rwar
d C
ATG
GC
AC
CA
CC
AG
CA
TCT
130
Rev
erse
A
TCC
GC
GTG
TTTC
TTTT
CG
A
Prob
e C
GC
CTG
CA
AG
TCC
TAA
GA
CG
CC
A
Neo
spor
a ca
ninu
m
NC
5 ge
ne
Forw
ard
GG
GA
TAC
GTG
GTT
TGTG
GTT
AG
Th
is st
udy
Rev
erse
C
AC
AG
AA
CA
CTG
AA
CTC
TCG
ATA
AG
Prob
e TC
AC
GTT
GA
AA
TCA
GC
CTG
CG
TCA
Sarc
ocys
tis c
ruzi
18
s rib
osom
al R
NA
gen
e Fo
rwar
d TC
TGC
TGG
AA
GC
AA
TCA
GTC
10
9
Rev
erse
TT
GA
AG
CA
GG
CTT
ATT
GC
CT
Prob
e A
CC
CA
TCTA
TATT
GG
GA
TAA
TAC
CG
TTA
CT
Toxo
plas
ma
gond
ii P3
0 ge
ne
Forw
ard
GC
CTC
ATC
GG
TCG
TCA
ATA
A
This
stud
y
Rev
erse
G
TCA
TTG
TAG
TGG
GTC
CTT
CC
Prob
e A
GC
AC
TCTT
GG
TCC
TGTC
AA
GTT
GT
Tritr
icho
mon
as fo
etus
5.
8S ri
boso
mal
RN
A G
ENE
Forw
ard
GC
GG
CTG
GA
TTA
GC
TTTC
TTT
159
Rev
erse
A
TGC
AC
ATT
GC
GC
GC
C
Prob
e A
CA
AG
TTC
GA
TCTT
TG
Bov
ine
para
influ
enza
viru
s 3
mat
rix (M
) pro
tein
Fo
rwar
d TG
TCTT
CC
AC
TAG
ATA
GA
GG
GA
TAA
AA
TT
86
Rev
erse
G
CA
ATG
ATA
AC
AA
TGC
CA
TGG
A
Prob
e A
CA
GC
AA
TTG
GA
TCA
ATA
A
Tab
le 2
.1. C
ontin
ued
53
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Influ
enza
D v
irus
PB1
Forw
ard
CA
GC
TGC
GA
TGTC
TGTC
ATA
AG
86
Rev
erse
A
CA
AA
TTC
GC
AG
GG
CC
ATT
A
Prob
e A
ATG
GA
CTT
TCTC
CTG
GG
AC
TGC
T
Bov
ine
rhin
itis A
viru
s 3D
pol
Forw
ard
CA
CC
TGA
AC
TATG
GA
CTT
GG
86
Rev
erse
C
AC
GG
CC
TCA
ATC
ATC
TG
Prob
e G
AC
GTG
GA
CTG
GC
AC
CA
GTT
TGC
Bov
ine
rhin
itis B
viru
s 3D
pol
Forw
ard
AA
CG
CG
ATT
GTG
TCC
TAG
GG
86
Rev
erse
G
CC
AC
TGA
GG
TTA
GC
TTC
TC
Prob
e C
TGTC
CTT
TGC
AC
GG
CG
TGG
Bov
ine
resp
irato
ry sy
ncyt
ial v
irus
Nuc
leoc
apsi
d Fo
rwar
d G
CA
ATG
CTG
CA
GG
AC
TAG
GTA
TAA
T 86
Rev
erse
A
CA
CTG
TAA
TTG
ATG
AC
CC
CA
TTC
T
Prob
e A
CC
AA
GA
CTT
GTA
TGA
TGC
TGC
CA
AA
GC
A
Bov
ine
aden
oviru
s 3
Hex
on
Forw
ard
ATT
AC
CA
GC
GTC
AA
CC
TCTA
C
86
Rev
erse
C
CG
CC
GA
GA
GA
TAG
TCA
TTA
AA
Prob
e TC
CA
CTT
TGG
AA
GC
TATG
CTC
CG
C
Man
nhei
mia
hae
mol
ytic
a so
dA
Forw
ard
ATT
AG
TGG
GTT
GTC
CTG
GTT
AG
86
Rev
erse
G
CG
TGA
TTTC
GG
TTC
AG
TTG
Prob
e C
TGA
AC
CA
AC
AC
GA
GTA
GTC
GC
TGC
Paste
urel
la m
ulto
cida
km
t-1
Forw
ard
GG
GC
TTG
TCG
GTA
GTC
TTT
86
Rev
erse
C
GG
CA
AA
TAA
CA
ATA
AG
CTG
AG
TA
Prob
e C
GG
CG
CA
AC
TGA
TTG
GA
CG
TTA
TT
His
toph
illus
som
ni
16S-
rRN
A
Forw
ard
AA
GG
CC
TTC
GG
GTT
GTA
AA
G
86
Rev
erse
C
CG
GTG
CTT
CTT
CTG
TGA
TTA
T
Prob
e C
GG
TGA
TGA
GG
AA
GG
CG
ATT
AG
True
pere
lla p
yoge
nes
plo-
Pyol
ysin
Fo
rwar
d A
TCA
AC
AA
TCC
CA
CG
AA
GA
G
86
Rev
erse
TT
GC
AG
CA
TGG
TCA
GG
ATA
C
Prob
e TC
GA
CG
GTT
GG
ATT
CA
GC
GC
AA
TA
Tab
le 2
.1. C
ontin
ued
54
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Myc
opla
sma
bovi
s op
pD
Forw
ard
TCA
AG
GA
AC
CC
CA
CC
AG
AT
86
Rev
erse
A
GG
CA
AA
GTC
ATT
TCTA
GG
TGC
AA
Prob
e TG
GC
AA
AC
TTA
CC
TATC
GG
TGA
CCC
T
Ure
apla
sma
dive
rsum
16
S-rR
NA
Fo
rwar
d C
ATT
AA
ATG
ATG
TGC
CTG
GG
TAG
TAC
61
2 86
Rev
erse
C
CCC
GTC
AA
TTC
CG
TTTG
Prob
e TT
CG
CA
AG
AA
TGA
AA
C
β-ac
tin
Act
in
Forw
ard
AG
CG
CA
AG
TAC
TCC
GTG
TG
156
Rev
erse
C
GG
AC
TCA
TCG
TAC
TCC
TGC
TT
Prob
e TC
GC
TGTC
CA
CC
TTC
CA
GC
AG
ATG
T
Tab
le 2
.1. C
ontin
ued
* Sim
bu g
roup
: Dou
glas
viru
s, Sa
thup
eri v
irus,
Sham
onda
viru
s, Sc
hmal
lenb
erg
viru
s
55
Table 2.2. Summary of information about the samples included in this study
Farm identifier Type of farm Inside/outside of farm Type of sample No. of samples
A Dairy Outside Gadfly 6
Inside Spider 1
Inside Mosquito 3
Inside Fly 14
Outside Feces of bird 7
Inside Intestine contents of rodent 1
B Dairy Inside Fly 14
Inside Cockroach 1
Outside Gadfly 2
Inside Gadfly 2
Inside Feces of bird 5
Outside Feces of bird 2
Inside Intestine contents of rodent 1
C Dairy Inside Fly 2
D Dairy Inside Fly 1
E Meat Inside Fly 2
Inside Spider 1
Inside Unidentified arthropod 1
Outside Gadfly 2
Outside Feces of rodent 2
Inside Feces of rodent 2
F Meat Inside Fly 2
Outside Gadfly 2
Outside Fly 2
G Meat Inside Fly 1
Outside Fly 1
Inside Feces of rodent 1
No information Feces of rodent 1
H Meat Inside Fly 1
Outside Fly 1
Outside Gadfly 3
Outside Fly 1
56
Farm identifier Type of farm Inside/outside of farm Type of sample No. of samples
J Meat Inside Fly 2
Outside Fly 1
I Meat Inside Fly 1
K Meat Inside Fly 1
Outside Fly 1
L Meat Inside Fly 1
M Meat Inside Fly 2
Inside Feces of rodent 1
Outside Feces of rodent 2
Inside Gadfly 1
N Meat Inside Fly 1
O Meat Inside Fly 1
P Meat Inside Fly 1
Outside Fly 1
Q Meat Inside Fly 2
R Meat Inside Fly 1
Outside Fly 1
No information Feces of rodent 2
S Meat Inside Fly 1
T Meat Inside Fly 1
Outside Fly 1
U Meat Inside Fly 1
Outside Fly 1
Inside Feces of rodent 1
Table 2.2. Continued
57
Tab
le 2
.3. P
oolin
g m
etho
d us
ed fo
r the
ext
ract
ed n
ucle
ic a
cids
obt
aine
d fro
m v
ecto
rs sa
mpl
es
Vec
tor
Num
ber o
f Sa
mpl
es
Pool
1
Pool
2
Pool
3
Pool
4
Pool
5
Pool
6
Pool
7
Pool
8
Pool
9
Pool
10
Fly
64
9 9
9 9
9 9
9 9
9 10
Fece
s of r
oden
t 14
7
7
Gad
fly
18
9 9
Fece
s of b
ird
14
7 7
Arth
ropo
d 7
7
Dat
a ar
e pr
esen
ted
as n
umbe
r of s
ampl
es
ente
d as
num
ber o
fs
58
Table 2.4. Results for the LOD of bovine abortive and respiratory disease complex pathogens obtained by using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems)
Pathogen Limit of detection (Copies/reaction)
Bovine Viral diarrheal Virus 100
Bovine coronavirus 100
Mammalian orthoreovirus 100
Bovine herpes virus-1 100
Salmonella Dublin 100
Salmonella Enteritidis 100
Salmonella Typhimurium 100
Bovine adenovirus 3 100
Bovine parainfluenza virus 3 100
Bovine respiratory syncytial virus 100
Influenza D virus 100
Bovine rhinitis A virus 100
Bovine rhinitis B virus 100
Bovine adenovirus 7 100
Mannheimia haemolytica 100
Neospora caninum 100
Campylobacter fetus subsp. venerealis 100
Toxoplasma gondii 100
Sarcocysits cruzi 100
Brucella abortus 100
Bluetongue virus 100
Akabane virus 100
Chuzan virus 100
Aino virus 100
Ibaraki virus 100
Simbu group 100
Chlamydophila abortus 100
59
Pathogen Limit of detection (Copies/reaction)
Tritrichomonas foetus 100
Listeria monocytogenes 100
Aspergillus spp. 100
Leptospira spp. 100
Pasteurella multocida 250
Histophillus somni 250
Trueperella pyogenes 250
Mycoplasma bovis 250
Ureaplasma diversum 250
Table 2.4. Continued
60
Table 2.5. Results obtained from pooled samples using Dembo-PCR (Abortogenic and respiratory diseases complex pathogens).
Type of victor No. of pool Samples/pool Dembo-PCR result
Fly
1 9 Negative
2 9 Negative
3 9 Negative
4 9 Negative
5 9 Negative
6 9 Negative
7 9 Negative
8 9 Negative
9 9 Negative
10 10 Negative
Feces of rodent 1 7 Negative
2 7 Negative
Gadfly 1 9 Negative
2 9 Negative
Feces of bird 1 7 Negative
2 7 Negative
Arthropod 1 7 Neospora Caninum
61
A B
Fig. 2.1. Gel images showing the detection of N. caninum by using nested PCR.
(A); Gel image of first PCR. (B); Gel image of second PCR. (M); 100 bp leader.
(1); N. caninum positive sample. (2); Negative control.
250 bp 500 bp
M 1 2 M 1 2
62
CHAPTER THREE
«Screening of Nasal and Fecal Samples from Goats for Detection of Infectious
Agents of Abortion, Diarrhea, and Respiratory Disease Complex by
Dembo-PCR »
63
III.1. Introduction:
Breeding of goats has become an important part of animal industry worldwide
for production of milk, meat, skin, and wool [99]. Livestock owners prefer to share
grazing between goats and cows to control internal parasites by reducing the parasite
load in the pasture, which goats efficiently eat forage that cattle eat less preferentially
[165]. However, the disease especially abortion, diarrhea and respiratory diseases
complex have been increased in goats, unlikely the infectious causative agents are rarely
investigated and various pathogens including viruses, bacteria and protozoa associated
with abortion, diarrhea and respiratory disease complex including BVDV, BHV-1,
BAdV, BAV3 and H. somni in cattle are involved in natural and experimental infections
in goats [16, 22, 80, 90, 99, 116, 119]. In addition, there are several shared infection
agents causing abortion, diarrhea, and respiratory diseases between cattle and goats
including BTV, salmonella spp., Leptospira spp., Campylobacter spp., E. coli, L.
monocytogenes, C. abortus, M. haemolytica, Trueperella pyogenes, C. Perfringens, U.
diversum, Sarcocystis spp. and T. gondii [5, 27, 41, 57, 74, 84, 95, 125, 150, 162]. On
the other hand, some of the infectious agents such as H. somni, which cause respiratory
disease in cattle was detected from clinical healthy goats [118], which indicate that the
goats could be the potential reservoirs of cattle infectious agents. Previous reports show
that same pathogens like BVDV, and foot and mouth disease virus (FMDV) could be
transmitted between cattle and goats [7, 16], however, may close contact and pasturing
goats with cattle increase the risk of infection of these pathogens.
Studies, which investigate and demonstrate the presence of a broad range of
cattle pathogens in goats are rather limited. In the previous studies, detection systems
for 19 bovine diarrheal agents, and 16 bovine respiratory disease complex agents by
using Dembo diarrhea-PCR and Dembo respiratory-PCR [86, 146], in addition to
Dembo abortion-PCR, as described in Chapter 1 were developed. These Dembo-PCR
64
systems have rapidity, high sensitivity, high specificity, and an excellent capacity for
simultaneous detection of all targeted infectious agents.
In this Chapter, I evaluated whether the infectious agents, causing abortion,
diarrhea and respiratory diseases in cattle, could be detected in goats by using the
Dembo-PCR system that particularly targeted a total of 50 pathogens including BVDV,
IBAV, Simbu group viruses (Schmallenberg virus, Douglas virus, Shamonda virus,
Sathuperi virus), AKAV, CHUV, , BHV-1, BTV, Aino virus, BEV, BCoV, BRAV,
BRBV, BRCV, Bovine torovirus, MRV, BPIV 3, BLV, BAdV 3, BAdV 7, BRSV, IDV,
BRAV, BRBV, M. haemolytica, H. somni, Trueperella pyogenes, Salmonella enterica
ser. Dublin, S. enterica ser. Typhimurium, S. enterica ser. Enteritidis, C. abortus, B.
abortus, C. fetus subsp. venerealis , L. monocytogenes, M. avium. Spp. Paratuberculosis,
C. Perfringens, Enterotoxogenic E. coli, M. bovis, U. diversum, Pasteurella multocida,
Leptospira spp., T. gondii, T. foetus, N. caninum, S. cruzi, E. Zuernii, E. bovis and
Aspergillus spp.
III.2. Material and methods
III.1.1. Primers and probes
A total of 47 primers and probes sets (for 50 Pathogens) were selected (Table
3.1). One set of primers and probe for β-actin was used as internal control of extraction
of nucleic acid [86, 146, 155]. All probes were indicated by the dye FAM (6-
carboxyfluorescein) at the 5´ end and the fluorescent dye TAMRA (6-
carboxytetramethylrhodamine) at the 3´ end. All primers and probes were purchased
from Sigma-Aldrich (St. Louis, MO, U.S.A.) and Integrated DNA Technologies.
III.1.2. Extraction of nucleic acids
From fecal samples of goats, bacterial, protozoal, and fungal DNAs were
extracted using QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) and from
nasal swab samples, bacterial, protozoal, and fungal DNAs were extracted using
65
QIAamp UCP pathogen mini Kit (Qiagen, Hilden, Germany). High Pure Viral Nucleic
Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used to extract viral
nucleic acid, according to the manufacturer’s instructions. The extracted nucleic acids
were stored at −80°C until use.
III.1.3. Real-time PCR amplification
All TaqMan real-time PCR assays were performed under the same reaction
condition used for Dembo diarrhea-PCR, Dembo respiratory-PCR and the Dembo
abortion-PCR [86, 146]. A One Step PrimeScript RT-PCR Kit (Perfect Real Time)
(TaKaRa Bio, Otsu, Japan) was used to detect viral RNA, and Premix Ex Taq (Perfect
Real Time) (TaKaRa Bio) was used to detect the viral, protozoal, fungal, and bacterial
DNAs. The real-time PCR assay was performed with the Applied Biosystems 7300
Real-Time PCR System (ABI 7300) for screening and with the LightCycler Nano
(Roche Diagnostics GmbH) for the validation of positive samples during screening. To
analyze the fluorescence data, the automatic analysis option was used in the LightCycler
Nano Software 1.1 (Roche Diagnostics GmbH) and the Applied Biosystems 7300 Real-
Time PCR software.
III.1.4. Clinical samples
A total of 50 samples, including 25 nasal swabs and 25 fecal swabs from 25
goats was collected from 1 farm in 2017 from Kumamoto prefecture in Japan. The
nucleic acids were extracted from each sample and evaluated in triplicated with Dembo-
PCR to screen a total of 50 bovine abortogenic, diarrheal, and respiratory disease
complex pathogens. When the Cq values were calculated with the algorithm in more
than two out of three runs, the samples were considered to be positive. All positive
samples were analyzed by conventional PCR (Table 3.2), to confirm the sequences of
detected pathogens by using cycle sequencing.
66
III.2. Results:
III.2.1. Analysis of clinical samples by using Dembo-PCR
All 50 samples were individually analyzed using Dembo-PCR. Fecal swab samples
were supposed to be negative for the targeted pathogens by using Dembo-PCR. All 25
nasal swab samples were positive for H. somni and 5 out of 25 samples were positive
for M. haemolytica by Dembo-PCR system (Table 3.3). The Dembo-PCR positive
results for M. haemolytica and H. somni were confirmed by conventional PCR (Fig. 3.1
and Fig. 3.2), and the sequences were obtained by cycle sequencing (data not shown).
III.3. Discussion
This study was the first evaluation of a highly sensitive, specific and rapid
pathogen detection system for simultaneous detection of wide-range of bovine
pathogens in goats.
I detected 2 infectious agents including H. somni and M. haemolytica in nasal
swab samples from 25 goats. However, fecal swab samples from same goats were
negative for the targeted pathogens by using Dembo-PCR
H. somni was detected in all 25 nasal swab samples from goats by using Dembo-
PCR. H. somni is a Gram-negative, facultative and fastidious pathogenic bacterium. It
had many previous names (Histophilus ovis, Haemophilus agni, Haemophilus somnus)
that were used in parallel, resulting in confusion. The name Histophilus somni was
suggested a few years ago [11]. For the first time, H. somni was isolated in Colorado,
US, in 1956, as an infectious agent of encephalitis in cattle [18], subsequently, as
causing meningoencephalitis and thromboembolic meningoencephalomyelitis [62],
pneumonia [10], otitis [36], and mastitis [70]. In sheep, H. somni causes orchitis,
epididymitis, and mastitis [116], abortion [105], synovitis, meningoencephalitis [119],
septicemia, and pneumonia [86]. There are only 2 reports of identification of H. somni
in healthy goats, however; there is no information about clinical infection by this
67
pathogen in goats [80, 118]. In one previous study, H. somni was isolated only from
genital mucous membranes of those goats, which were kept together with sheep [80],
and the presence of H. somni on goat genital mucous membranes was in correlation with
the oestrus season and the intensity of sheep contact [80]. However, the nasal discharge
samples were negative for H. somni [80].
I detected H. somni from the nasal cavity of all 25 goats by Dembo-PCR, which
only one goat showed the nasal discharges and other goats were clinically healthy. As
far as I know, this is the first report of detection of H. somni in goats in Japan. However,
in a previous study, H. somni was detected in nasal swab samples from cattle by Dembo-
PCR [86].
M. haemolytica was detected in 5 out of 25 nasal swab samples from goats. The
goats with positive results were in good health based on physical appearance. All goats
were in an exhibition farm as herd goats. A previous study showed that, herd goats had
a higher prevalence of Mannheimia spp. isolations than pack goats [35]. M. haemolytica
is a Gram-negative organism, which closely associated with the BRDC [86] and it is
one of the important infectious agents of pneumonia in cattle, sheep, and goats [160].
Identification of M. haemolytica with bacteriological methods is expected to be difficult
in some situations such as antibiotic treatment, frozen material, autolytic material and
others [160]. Dembo-PCR system was already developed and validated for detection of
M. haemolytica in cattle [86]. However, to my knowledge, this is the first demonstration
of M. haemolytica in goats by using a Taq-Man real-time PCR based tool. I confirmed
all 5 M. haemolytica positive results by conventional PCR (Fig .3.1), and the sequences
were obtained by cycle sequencing (data not shown).
68
In conclusion, the present and the previous studies [86, 146] demonstrated that
Dembo-PCR was a highly sensitive, specific and rapid detection technique for the
detection of a wide-range of pathogens in various samples obtained from different
animals, under the same reaction conditions.
69
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Ain
o vi
rus
M p
olyp
rote
in
Forw
ard
AG
CA
AA
TCC
CA
TTG
CG
TGA
Th
is st
udy
Rev
erse
C
AG
AC
TTC
TGC
TGG
CA
CA
TTA
Prob
e A
GG
GA
CA
AC
TGG
CTC
TCG
CT
Aka
bane
viru
s S
segm
ent
Forw
ard
TCA
AC
CA
GA
AG
AA
GG
CC
AA
GA
T 13
7
Rev
erse
G
GG
AA
AA
TGG
TTA
TTA
AC
CA
CTG
TAA
A
Prob
e TT
AC
ATA
AG
AC
GC
CA
CA
AC
CA
Blu
eton
gue
viru
s N
S3 g
ene
Forw
ard
AA
ATM
TTG
GA
YA
AA
GC
RA
TGTC
AA
A
154
Rev
erse
C
TYA
CR
TCA
TCA
CG
AA
AC
GC
T
Prob
e A
AR
GC
TGC
ATT
CG
CA
TCG
TAC
GC
Chu
zan
viru
s V
P7 g
ene
Forw
ard
TGA
TCG
AA
CG
CC
AA
CA
CTT
Th
is st
udy
Rev
erse
G
GC
AA
TCC
AA
CC
CTC
ATA
CA
Prob
e TA
TCA
CC
AC
AA
TGG
CA
TGC
ATT
GC
G
Ibar
aki v
irus
VP3
gen
e Fo
rwar
d TA
CA
GC
GG
GA
CC
TAG
GTT
TA
This
stud
y
Rev
erse
G
TTC
TCC
CG
TTG
GA
CC
ATA
TT
Prob
e TG
GC
AC
GA
CA
GC
TTG
ATA
TTG
CC
T
Sim
bu g
roup
* N
, NSs
gen
e, S
segm
ent
Forw
ard
TGA
AG
ATG
TAC
CA
CA
AC
GG
AA
T Th
is st
udy
Rev
erse
G
AG
GA
AG
AA
GA
CTC
TAG
CA
AC
AC
Prob
e A
CC
TCC
GG
GTT
AA
ATG
TAG
CTG
C
Aspe
rgill
us sp
p.
18S
ribos
omal
RN
A g
ene
Forw
ard
GC
CC
GC
CG
TTTC
GA
C
98
Rev
erse
C
CG
TTG
TTG
AA
AG
TTTT
AA
CTG
ATT
AC
Prob
e C
CCG
CC
GA
AG
AC
CC
CA
AC
ATG
Bruc
ella
abo
rtus
omp2
a ge
ne
Forw
ard
GC
GG
CTT
TTC
TATC
AC
GG
TATT
C
122
Rev
erse
C
ATG
CG
CTA
TGA
TCTG
GTT
AC
G
Prob
e C
GC
TCA
TGC
TCG
CC
AG
AC
TTC
AA
TG
Cam
pylo
bact
er fe
tus s
ubsp
. Ven
erea
lis
nahE
gen
e Fo
rwar
d TT
CA
AA
AG
CTC
TTG
GG
GTT
AC
58
Rev
erse
A
AA
GC
CTT
GTT
TAG
AA
CA
ATA
TAA
CTC
Prob
e A
CTC
GTG
GTG
GA
GA
GC
GTA
G
Tab
le 3
.1. I
nfor
mat
ion
on a
ll pr
imer
s and
pro
bes u
sed
in c
urre
nt st
udy
70
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Chl
amyd
ophi
la a
bortu
s
om
pA g
ene
Forw
ard
G
CA
AC
TGA
CA
CTA
AG
TCG
GC
TAC
A
11
7
Rev
erse
A
CA
AG
CA
TGTT
CA
ATC
GA
TAA
GA
GA
Prob
e TA
AA
TAC
CA
CG
AA
TGG
CA
AG
TTG
GTT
TAG
CG
Lept
ospi
ra sp
p.
lipL3
2 ge
ne
Forw
ard
AA
GC
ATT
AC
CG
CTT
GTG
GTG
13
9
Rev
erse
G
AA
CTC
CC
ATT
TCA
GC
GA
T
Prob
e A
AA
GC
CA
GG
AC
AA
GC
GC
CG
List
eria
mon
ocyt
ogen
es
iap
gene
Fo
rwar
d C
ATG
GC
AC
CA
CC
AG
CA
TCT
130
Rev
erse
A
TCC
GC
GTG
TTTC
TTTT
CG
A
Prob
e C
GC
CTG
CA
AG
TCC
TAA
GA
CG
CC
A
Neo
spor
a ca
ninu
m
NC
5 ge
ne
Forw
ard
GG
GA
TAC
GTG
GTT
TGTG
GTT
AG
Th
is st
udy
Rev
erse
C
AC
AG
AA
CA
CTG
AA
CTC
TCG
ATA
AG
Prob
e TC
AC
GTT
GA
AA
TCA
GC
CTG
CG
TCA
Sarc
ocys
tis c
ruzi
18
s rib
osom
al R
NA
gen
e Fo
rwar
d TC
TGC
TGG
AA
GC
AA
TCA
GTC
10
9
Rev
erse
TT
GA
AG
CA
GG
CTT
ATT
GC
CT
Prob
e A
CC
CA
TCTA
TATT
GG
GA
TAA
TAC
CG
TTA
CT
Toxo
plas
ma
gond
ii P3
0 ge
ne
Forw
ard
GC
CTC
ATC
GG
TCG
TCA
ATA
A
This
stud
y
Rev
erse
G
TCA
TTG
TAG
TGG
GTC
CTT
CC
Prob
e A
GC
AC
TCTT
GG
TCC
TGTC
AA
GTT
GT
Tritr
icho
mon
as fo
etus
5.
8S ri
boso
mal
RN
A G
ENE
Forw
ard
GC
GG
CTG
GA
TTA
GC
TTTC
TTT
159
Rev
erse
A
TGC
AC
ATT
GC
GC
GC
C
Prob
e A
CA
AG
TTC
GA
TCTT
TG
Bov
ine
ente
rovi
rus
5′U
TR
Forw
ard
GC
CG
TGA
ATG
CTG
CTA
ATC
C
146
Rev
erse
G
TAG
TCTG
TTC
CG
CC
TCC
AC
CT
Prob
e C
GC
AC
AA
TCC
AG
TGTT
GC
TAC
GTC
GTA
AC
Tab
le 3
.1. C
ontin
ued
71
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Gro
up A
rota
viru
s V
P6
Forw
ard
AC
TCC
AA
TGTA
AG
TGA
TCTA
ATT
C
146
Rev
erse
G
AG
TTG
TTC
CA
AG
TAA
TCC
AA
A
Prob
e A
CC
AA
TTC
CTC
CA
GTT
TGG
AA
YTC
ATT
YC
C
Gro
up B
rota
viru
s V
P6
Forw
ard
TGG
CA
GG
TGG
TCA
GG
TAA
TAA
14
6
Rev
erse
A
CA
CC
AC
AC
GTT
CTA
GC
TTTC
AG
Prob
e G
TCA
CA
TGTG
TCTC
AG
GC
ATG
GA
AG
C
Gro
up C
rota
viru
s V
P6
Forw
ard
GC
CA
ATA
CG
AG
AA
GG
GA
TTC
14
6
Rev
erse
TC
TTC
AC
GG
ATG
CA
AC
TAG
C
Prob
e C
CA
GG
ATT
TCC
ATG
GG
AA
CA
GA
CG
T
Bov
ine
coro
navi
rus
Nuc
leoc
apsi
d Fo
rwar
d C
TGG
AA
GTT
GG
TGG
AG
TT
146
Rev
erse
A
TTA
TCG
GC
CTA
AC
ATA
CA
TC
Prob
e C
CTT
CA
TATC
TATA
CA
CA
TCA
AG
TTG
TT
Bov
ine
toro
viru
s N
ucle
ocap
sid
Forw
ard
CG
TATT
CA
AA
AC
CA
AA
GA
CG
TG
146
Rev
erse
G
TGC
AG
TCTC
ATT
TGC
CA
TC
Prob
e C
CA
GC
AG
TCA
CTA
TCTT
TGC
CA
TTTG
A
Mam
mal
ian
orth
oreo
viru
s λ3
Fo
rwar
d TC
GA
TATC
GG
GA
ATG
CA
G
146
Rev
erse
C
TGA
CG
GG
AA
AG
TGG
TRG
TCA
Prob
e A
TGA
TCC
AG
CA
TCTA
TCG
AA
AC
TRTA
TAA
AC
G
Bov
ine
leuk
emia
viru
s Po
l Fo
rwar
d C
CTC
AA
TTC
CC
TTTA
AA
CTA
14
6
Rev
erse
G
TAC
CG
GG
AA
GA
CTG
GA
TTA
Prob
e G
AA
CG
CC
TCC
AG
GC
CC
TTC
A
Bov
ine
aden
oviru
s 7
Hex
on
Forw
ard
CR
AG
GG
AA
TAY
YTG
TCTG
AA
AA
TC
146
Rev
erse
A
AG
GA
TCTC
TAA
ATT
TYTC
TCCA
AG
A
Prob
e TT
CA
TCW
CTG
CC
AC
WC
AA
AG
CTT
TTTT
Salm
onel
la D
ublin
V
agC
Fo
rwar
d G
GG
TGA
GC
GA
GC
TGG
AA
A
146
Rev
erse
C
GC
CA
TAA
AG
TCC
GG
GTC
A
Prob
e TT
TTTC
GA
GC
TGC
GC
GA
AC
GA
GC
Tab
le 3
.1. C
ontin
ued
72
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Salm
onel
la E
nter
itidi
s Se
fA
Forw
ard
GG
TAA
AG
GG
GC
TTC
GG
TATC
14
6
Rev
erse
TA
TTG
GC
TCC
CTG
AA
TAC
GC
Prob
e TG
GTG
GTG
TAG
CC
AC
TGTC
CC
GT
Salm
onel
la T
yphi
mur
ium
Fi
c Fo
rwar
d TG
CA
GA
AA
ATT
GA
TGC
TGC
T 14
6
Rev
erse
TT
GC
CC
AG
GTT
GG
TAA
TAG
C
Prob
e A
CC
TGG
GTG
CG
GTA
CA
GA
AC
CG
T
Myc
obac
teri
um a
vium
. Ssp
. Pa
ratu
berc
ulos
is
IS90
0 Fo
rwar
d C
AG
CG
GC
TGC
TTTA
TATT
CC
14
6
Rev
erse
G
CA
GA
GG
CTG
CA
AG
TCG
T
Prob
e A
AG
AC
CG
AC
GC
CA
AA
GA
CG
CTG
CG
A
Clo
strid
ium
per
fring
ens
Cpb
Fo
rwar
d A
TTTC
ATT
AG
TTA
TAG
TTA
GTT
CA
C
146
Rev
erse
TT
ATA
GTA
GTA
GTT
TTG
CC
TATA
TC
Prob
e A
AC
GG
ATG
CC
TATT
ATC
AC
CA
AC
T
Ente
roto
xoge
nic
Esch
eric
hia
coli
K99
Fo
rwar
d G
CTA
TTA
GTG
GTC
ATG
GC
AC
TGTA
G
146
Rev
erse
TT
TGTT
TTG
GC
TAG
GC
AG
TCA
TTA
Prob
e A
TTTT
AA
AC
TAA
AA
CC
AG
CG
CC
CG
GC
A
Eim
eria
zuer
nii/b
ovis*
* IT
S1
Forw
ard
TGTC
TAY
AC
AC
AC
TMC
ATC
CA
AC
14
6
Rev
erse
C
T G
AC
CA
CA
GTG
TTG
GA
AA
TGC
Prob
e TG
GC
CTG
TTG
TGG
ATA
GTT
AC
TG (Z
uern
ii)
Prob
e G
CC
TTA
TGG
ATA
GTT
AG
TGC
TCC
(bov
is)
Bov
ine
para
influ
enza
viru
s 3
mat
rix (M
) pro
tein
Fo
rwar
d TG
TCTT
CC
AC
TAG
ATA
GA
GG
GA
TAA
AA
TT
86
Rev
erse
G
CA
ATG
ATA
AC
AA
TGC
CA
TGG
A
Prob
e A
CA
GC
AA
TTG
GA
TCA
ATA
A
Bov
ine
resp
irato
ry sy
ncyt
ial v
irus
Nuc
leoc
apsi
d Fo
rwar
d G
CA
ATG
CTG
CA
GG
AC
TAG
GTA
TAA
T 86
Rev
erse
A
CA
CTG
TAA
TTG
ATG
AC
CC
CA
TTC
T
Prob
e A
CC
AA
GA
CTT
GTA
TGA
TGC
TGC
CA
AA
GC
A
Tab
le 3
.1. C
ontin
ued
73
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Influ
enza
D v
irus
PB1
Forw
ard
CA
GC
TGC
GA
TGTC
TGTC
ATA
AG
86
Rev
erse
A
CA
AA
TTC
GC
AG
GG
CC
ATT
A
Prob
e A
ATG
GA
CTT
TCTC
CTG
GG
AC
TGC
T
Bov
ine
rhin
itis A
viru
s 3D
pol
Forw
ard
CA
CC
TGA
AC
TATG
GA
CTT
GG
86
Rev
erse
C
AC
GG
CC
TCA
ATC
ATC
TG
Prob
e G
AC
GTG
GA
CTG
GC
AC
CA
GTT
TGC
Bov
ine
rhin
itis B
viru
s 3D
pol
Forw
ard
AA
CG
CG
ATT
GTG
TCC
TAG
GG
86
Rev
erse
G
CC
AC
TGA
GG
TTA
GC
TTC
TC
Prob
e C
TGTC
CTT
TGC
AC
GG
CG
TGG
Bov
ine
herp
esvi
rus 1
gE
Fo
rwar
d C
AA
TAA
CA
GC
GTA
GA
CC
TGG
TC
86
Rev
erse
G
CTG
TAG
TCC
CA
AG
CTT
CC
AC
Prob
e TG
CG
GC
CTC
CG
GG
CTT
TAC
GTC
T
Bov
ine
aden
oviru
s 3
Hex
on
Forw
ard
ATT
AC
CA
GC
GTC
AA
CC
TCTA
C
86
Rev
erse
C
CG
CC
GA
GA
GA
TAG
TCA
TTA
AA
Prob
e TC
CA
CTT
TGG
AA
GC
TATG
CTC
CG
C
Man
nhei
mia
hae
mol
ytic
a so
dA
Forw
ard
ATT
AG
TGG
GTT
GTC
CTG
GTT
AG
86
Rev
erse
G
CG
TGA
TTTC
GG
TTC
AG
TTG
Prob
e C
TGA
AC
CA
AC
AC
GA
GTA
GTC
GC
TGC
Paste
urel
la m
ulto
cida
km
t-1
Forw
ard
GG
GC
TTG
TCG
GTA
GTC
TTT
86
Rev
erse
C
GG
CA
AA
TAA
CA
ATA
AG
CTG
AG
TA
Prob
e C
GG
CG
CA
AC
TGA
TTG
GA
CG
TTA
TT
His
toph
illus
som
ni
16S-
rRN
A
Forw
ard
AA
GG
CC
TTC
GG
GTT
GTA
AA
G
86
Rev
erse
C
CG
GTG
CTT
CTT
CTG
TGA
TTA
T
Prob
e C
GG
TGA
TGA
GG
AA
GG
CG
ATT
AG
True
pere
lla p
yoge
nes
plo-
Pyol
ysin
Fo
rwar
d A
TCA
AC
AA
TCC
CA
CG
AA
GA
G
86
Rev
erse
TT
GC
AG
CA
TGG
TCA
GG
ATA
C
Prob
e TC
GA
CG
GTT
GG
ATT
CA
GC
GC
AA
TA
Tab
le 3
.1. C
ontin
ued
74
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
/Pro
be se
quen
ce 5
'-3' (
FAM
-TA
MR
A)
Ref
eren
ce N
o.
Myc
opla
sma
bovi
s op
pD
Forw
ard
TCA
AG
GA
AC
CC
CA
CC
AG
AT
86
Rev
erse
A
GG
CA
AA
GTC
ATT
TCTA
GG
TGC
AA
Prob
e TG
GC
AA
AC
TTA
CC
TATC
GG
TGA
CCC
T
Ure
apla
sma
dive
rsum
16
S-rR
NA
Fo
rwar
d C
ATT
AA
ATG
ATG
TGC
CTG
GG
TAG
TAC
61
2 86
Rev
erse
C
CCC
GTC
AA
TTC
CG
TTTG
Prob
e TT
CG
CA
AG
AA
TGA
AA
C
Bov
ine
vira
l dia
rrhea
viru
s 5′
UTR
Fo
rwar
d G
GG
NA
GTC
GTC
AR
TGG
TTC
G
146
Rev
erse
G
TGC
CA
TGTA
CA
GC
AG
AG
WTT
TT
Prob
e C
CA
YG
TGG
AC
GA
GG
GC
AY
GC
β-ac
tin
Act
in
Forw
ard
AG
CG
CA
AG
TAC
TCC
GTG
TG
156
Rev
erse
C
GG
AC
TCA
TCG
TAC
TCC
TGC
TT
Prob
e TC
GC
TGTC
CA
CC
TTC
CA
GC
AG
ATG
T
Tab
le 3
.1. C
ontin
ued
* Sim
bu g
roup
: Dou
glas
viru
s, Sa
thup
eri v
irus,
Sham
onda
viru
s, Sc
hmal
lenb
erg
viru
s
**Sa
me
prim
ers,
but d
iffer
ent p
robe
s
75
Targ
et p
atho
gen
Targ
et g
ene
Dire
ctio
n Pr
imer
sequ
ence
5' -
3'
PCR
pro
duct
size
R
efer
ence
No.
Man
nhei
mia
hae
mol
ytic
a 16
S-rR
NA
Fo
rwar
d G
TGC
CG
GG
AA
ATC
AA
TCG
CT
144b
p 86
R
ever
se
GC
CA
TAA
ATA
AG
CA
GG
GC
TATG
TGG
His
toph
illus
Som
ni
16
S-rR
NA
Fo
rwar
d CATTTCAGACTGGGTGACTAGAG
37
3bp
This
stud
y R
ever
se
CGGCTTCTTAGGATGTCAAGAG
Tab
le 3
.2. I
nfor
mat
ion
on c
onve
ctio
nal P
CR
prim
ers u
sed
in c
urre
nt st
udy
stud
y
76
Sample No. Sample type Symptom Detected pathogen
1 Nasal swab Nasal discharge H.somni + M.haemolytica
2 Nasal swab Healthy H.somni + M.haemolytica
3 Nasal swab Healthy H.somni
4 Nasal swab Healthy H.somni + M.haemolytica
5 Nasal swab Healthy H.somni
6 Nasal swab Healthy H.somni
7 Nasal swab Healthy H.somni
8 Nasal swab Healthy H.somni
9 Nasal swab Healthy H.somni + M.haemolytica
10 Nasal swab Healthy H.somni
11 Nasal swab Healthy H.somni
12 Nasal swab Healthy H.somni
13 Nasal swab Healthy H.somni + M.haemolytica
14 Nasal swab Healthy H.somni
15 Nasal swab Healthy H.somni
16 Nasal swab Healthy H.somni
17 Nasal swab Healthy H.somni
18 Nasal swab Healthy H.somni
19 Nasal swab Healthy H.somni
20 Nasal swab Healthy H.somni
21 Nasal swab Healthy H.somni
22 Nasal swab Healthy H.somni
23 Nasal swab Healthy H.somni
24 Nasal swab Healthy H.somni
25 Nasal swab Healthy H.somni
H. somni; Histophilus somni, M. haemolytica; Mannheimia haemolytica
Table 3.3. Positive results of clinical samples from goats using Demo-PCR
D
o- RPCR
77
Fig. 3.1 (A, B). Gel image showing the detection of M. haemolytica by using conventional PCR. (M); 100 bp leader. (1, 2, 3); M. haemolytica positive samples. (4); Negative control.
144bp
M 1 2 3 4
144bp
M 1 2 3 4
A
B
78
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 N M
M 1 2 3 4 5 6 7 8 9 10 11 N M
373bp
373bp
Fig. 3.2 (A, B). Gel image showing the detection of H. somni by using conventional PCR. (M); 100 bp leader. (1-14); H. somni positive samples. (N); Negative control.
A
B
79
« General Conclusion»
80
In this dissertation, a simultaneous TaqMan real-time PCR system (Dembo
abortion-PCR) was developed for detection and differentiation of 24 cattle abortogenic
infectious agents. Subsequently, all 3 Dembo-PCR systems (Dembo abortion-PCR,
Dembo diarrhea-PCR and, Dembo respiratory-PCR) were combined to detect a total of
50 cattle abortogenic, diarrheal and respiratory disease complex infectious agents from
potential vectors and reservoirs such as flies, rodents, birds, arthropods and as well as
goats.
In Chapter one, I developed Dembo abortion-PCR for simultaneous detection of
24 cattle abortogenic infectious agents including 11 viruses, 8 bacteria, 4 protozoa, and
1 fungus. Sensitivity of Dembo abortion-PCR were validated using synthesized DNAs
of each target pathogens. Subsequently 22 blood samples from cattle and, 40 aborted
fetus samples from cattle and 2 aborted fetus samples form pigs were analyzed,
respectively. All samples from cattle including blood samples and aborted fetus samples
were negative for all of the targeted abortogenic pathogens. However, Akabane virus
was detected from both aborted fetus samples from pigs (Table 1.5).
In Chapter two, Dembo-PCR including Dembo abortion-PCR, and Dembo
respiratory-PCR were applied for detection of 31 bovine abortogenic, and respiratory
disease complex infectious agents in vectors and reservoirs such as birds, arthropods,
rodents and combined the results with Dembo diarrhea-PCR. A total of 117 samples
from vectors and reservoirs were collected and pooled (Table 2.3). All 15 pooled
samples were screened by Dembo respiratory- and abortion-PCR.
N. caninum was detected from the arthropod pooled sample (Tables 2.5), which
consisted of 7 different arthropods samples including 2 cockroaches, 2 spiders, and 3
unidentified arthropods. To clarify which arthropods were positive for N. caninum, all
7 arthropod samples were analyzed in the LightCycler Nano instrument, and found that,
the cockroach sample showed positive reaction exclusively.
81
In Chapter three, a total of 50 fecal and nasal swab samples from goats for
detection of all 50 previously mentioned cattle abortogenic, diarrheal and respiratory
disease complex infectious agents were screened by using the Dembo-PCR. This study
was the first evaluation of a highly sensitive, specific, and rapid pathogen detection
system for simultaneous detection of broad-range of cattle pathogens and cattle and
goats shared pathogens in goats.
Two infectious agents including H. somni and M. haemolytica were detected
from nasal swab samples of 25 goats (Table 3.3). However, I could not identify any of
the targeted pathogens by using Dembo-PCR from fecal swab samples.
Through these studies, I improved the Dembo-PCR as a wide-range
simultaneous pathogens detection system with high sensitivity, high specificity, and
rapidity. This system has the capacity to detect 50 bovine abortogenic, diarrheal and
respiratory disease complex infectious agents, simultaneously. Subsequently I applied
the Dembo-PCR to show the role of flies, rodents, arthropods, birds, and goats as
potential vectors and reservoirs of these 50 pathogens.
Dembo-PCR was developed for detection of cattle abortion, diarrhea and
respiratory diseases infectious agents. However; further studies are needed to develop
such systems for simultaneous detection of infectious agents of diseases in the urinary
system, nerve system, oral cavity, eyes, skin, and etc in cattle.
Dembo-PCR is a valuable system for differential diagnosis of infectious agents
of diseases with similar clinical signs. For example, Rinderpest, BVD, and Bovine
malignant catarrhal fever have similar clinical signs. Development of a Dembo-PCR
system for differential diagnosis of these diseases could be an excellent idea.
82
«Acknowledgements»
83
It is my great pleasure to acknowledge the following persons for their kind helps
and supports in my four years of doctoral course.
Firstly, I would like to express my sincere gratitude to my supervisor, Prof. Dr.
Tetsuya Mizutani, Research and Education Center for Prevention of Global Infectious
Diseases of Animal, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, for the continuous support of my PhD study
and related research, for his patience, motivation, and immense knowledge.
Subsequently, I am greatly indebted to my co-adviser, Assoc. Prof. Dr. Tsutomu
Omatsu, Research and Education Center for Prevention of Global Infectious Diseases
of Animal, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, for his valuable supports in all the time of
research and writing of the thesis.
Besides my Advisors, I would like to thank the rest of my thesis committee:
Prof. Dr. Tetsuo Asai, Prof. Dr. Shigeru Morikawa, Prof. Dr. Haruko Ogawa, Prof. Dr.
Tatsuya Furuichi, and Prof. Dr. Tetsuya Furuya for their insightful comments and
encouragements, but also for the hard questions, which incented me to widen my
research from various perspectives.
I am also grateful of the honest supports of Dr. Shinobu Tsuchiaka, who was a
nice friend and kind teacher for me.
A very special gratitude to all of our laboratory members for their patience and
support in overcoming numerous obstacles I have been facing through my research.
Last, but by no means least, I would like to thank my family, specially my father
Sayed Yahya and my mother Sediqa, for their true love and supports throughout my
life. I am also thankful of my wife, Tahmina Rahpaya, for her patience and supports
during my research, writing and laboratory working times in Japan.
84
And finally, I am grateful of the noble people of japan for providing me the full-
scholarship of this doctoral course. I am sure without their support reaching to this
achievement was not possible.
85
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