Title Studies on Improvement and Validation of …...BVD Bovine viral diarrhea BVDV Bovine viral diarrhea virus cDNA complementary DNA CHUV Chuzan virus Dembo-PCR Detection system

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

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GN

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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

















18 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






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


Aino virus

BVDV


Cq

valu

e

Chuzan virus

Cq v

alue


Ibarake virus

Cq

valu

e


Simbu Group viruses


Cq

valu

e

BHV-1


Cq

valu

e

Bluetongue virus


Cq

valu

e

37

Fig. 1.2 (B). Standard curves using synthesized DNA

Cq

valu

e


Brucella abortus Chlamydophila abortus

Cq

valu

e


Champylobacter fetus

Cq

valu

e


Leptospira spp.


Cq

valu

e

Listeria monocytogenes

Cq

valu

e


Salmonella enetritidis


Cq

valu

e

Salmonella Typhimurium

Cq

valu

e


Salmonella Dublin


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


Cq

valu

e

Toxoplasma gondii

Cq

valu

e


Neospora caninum

Cq

valu

e


Tritrichomonas fetus

Cq

valu

e


Aspergillus spp.

Cq

valu

e


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

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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


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


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



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


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























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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