Arfan Ahmad - prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1326/1/2438S.pdf · ARFAN AHMAD, have been found satisfactory and recommend that it be processed for the evaluation

I

COMPARISON OF DIAGNOSTIC APPROACHES FOR

THE DETECTION OF BOVINE VIRAL DIARRHEA

PERSISTENCY IN DAIRY HERDS

By

Arfan Ahmad

(2007-VA-437)

A Thesis Submitted in the Partial Fulfillment of the Requirement for the Degree

Of

DOCTOR OF PHILOSOPHY

IN

MICROBIOLOGY

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,

LAHORE

2012

II

To

The Controller of Examinations,

University of Veterinary and Animal Sciences,

Lahore.

We, the Supervisory Committee, certify that the contents and form of the thesis, submitted by

ARFAN AHMAD, have been found satisfactory and recommend that it be processed for the

evaluation by the External Examiner(s) for the award of the degree.

SUPERVISOR_______________________________________

Prof. Dr. Masood Rabbani (Izaz-i Fazeelat)

MEMBER _______________________________________

Prof. Dr. Khushi Muhammad

MEMBER ______________________________________

Prof. Dr. Rana Muhammad Younis

III

I would like to pay all my praises and humblest thank to Most Gracious, Merciful and

Almighty “ALLAH” who bestowed me with potential and ability to contribute some material to

the existing knowledge in the field of Microbiology and made everything possible for me to

complete my PhD Degree. I offer my humblest thanks from the core of my heart to the HOLY

PROPHET “MUHAMMAD” (S.A.W.) who is forever a torch of guidance and knowledge for

humanity as a whole.

I deem it as my utmost pleasure to avail this opportunity to express deep sense of obligation to

my venerated Supervisor, Prof. Dr. Masood Rabbani (Izaz-i-Fazeelat), Director, University

Diagnostic Laboratory, members of my supervisory committee, Dr. Khushi Muhammad,

chairman, Department of Microbiology, UVAS, Lahore, Prof. Dr. Rana Muhammad Younis,

Principal, CVS, Jhang, Prof. Dr. Arnost Cepica, Dept.of Pathology & Microbiology, Atlantic

Veterinary College, Prince Edward University, Charlottetown, Canada, ,Toki Lab Technologist,

Supervisor Lahore for their skillful guidance and inspiring attitude which made it very easy for

me to undertake this work.

Indeed, I would like to pay my regard to my all colleagues specially, Dr Shawn

McKenna, Dr. Edward, Kathene Jones, Diane, Rita, Amy Ortiz, Prof Dr. Tahir yaqub, Dr. Aftab,

Dr. Jawad, Dr. Imran, Dr. Ali Ahmad, Dr. Zubair Shabbir, Dr. Fariha, Dr. Mushtaq, Dr.

Khurram, Dr. Zia, Dr. afzal, Dr.Ghazanfar, Azeem, Atta ul islam for his dedicated cooperation,

all the ways tills the completion of my thesis. I find no word to express my gratitude to my

parents, brothers, sisters, my wife and sons Rayan and Adeen for their good wishes for my health

and success.

At the end, as is customary, that all mistakes left uncorrected are entirely mine.

ARFAN AHMAD

IV

DEDICATE THE FRUIT OF THIS HUMBLE EFFORT TO

HOLY PROPHET (SAW)

THE GREAT SOCIAL REFORMER

MY PARENTS

WHO ALWAYS APPRECIATE AND PRAY FOR ME TO ACHIEVE

HIGHER GOALS OF LIFE

V

TABLE OF CONTENTS

PAGE NO Title

I Title of thesis

III Acknowledgements

IV Dedication

V Table of contents

VI List of tables

VIII List of Figures

Sr. No CHAPTERS PAGE NO

1.

Introduction 1

2.

Review of literature 5

3.

Materials and Methods 34

4.

Results 52

5.

Discussion 93

6.

Summary 109

7.

Literature Cited 112

8 Appendices 140

VI

LIST OF TABLES

TABLE

NO.

TITLE PAGE

NO

1 Test approaches 50

2 Oligonucleotide primers and TaqMan probes 50

3 ELISA Interpretation 44

4 ELISA Interpretation of suspected samples 45

5 Calculation of TCID50 62

6 Anti-BVDV-Serum neutralizing antibody titres in vaccinated herds 63

7 Anti-BVDV-Serum neutralizing antibody titres in non-vaccinated herds 63

8 GroupWise Distribution of anti-BVDV- Serum neutralizing antibody titres 64

9 Comparison of detection limits of Real time RT-PCR and AC-ELISA. 64

10

Comparative Suitability of ear notch biopsies and serum samples by Real

time RT-PCR for the detection of persistent infection

65

11

Overall comparative efficacy of ear notch biopsies and sera by Real time RT-

PCR for the detection of persistent infection

66

12 Prevalence of BVDV persistency 67

13 Virus isolation on ear notch biopsies 68

14 Antigen Capture ELISA on ear notch biopsies 68

15

Immunohistochemistry on ear notch biopsies using Alkaline phosphatase

Naphthol detection kit

69

16 Comparative efficacy of various diagnostic approaches for the detection of 69

VII

BVDV persistency using ear notch biopsies.

17 McNemar test 70

18 Chi-Square Test: VI and AC-ELISA 70

19 Chi-Square Test: VI and IHC 71

20 Chi-Square Test: VI and Real Time RT-PCR 71

VIII

LIST OF FIGURES

FIGURE

NO. TITLE PAGE

NO.

1 BVDV Classification 7

2 Pestivirus Structure 8

3 Genome Organization 8

4 MDBK Cells During Dividing and Confluency stage 36

5 Flow Chart Regarding Materials & Methods of the study 51

6 CPE in the 96 well Microtitration Plate 72

7 Overall Seropositivity and GroupWise Distribution of Antibodies 72

8(a) TaqMan Probe 1 Specificity 73

8(b) TaqMan Probe 2 Specificity. 74

8(c)

Agarose gel electrophoresis of amplified products obtained by probe

specificity reactions

75

9

Comparison of Ear notch biopsies and Sera by Real time RT-PCR for

the detection of PI animals

76

10(a)

Detection of BVDV-1 Genotype by Real time RT-PCR using ear

notch biopsies during first round of testing

77

10(b)

Confirmation of amplified products (BVDV-1 amplicons) through

Agarose Gel electrophoresis during first round of testing

78

11(a) Detection of BVDV-1 Genotype by Real time RT-PCR using ear 79

IX

notch biopsies during second round of testing

11(b)

Confirmation of amplified products (BVDV-1 amplicons) through

Agarose Gel electrophoresis during second round of testing

80

12

Detection of BVDV-2 Genotypes by Real time RT-PCR using ear

notch biopsies during first round of testing

81

13

Detection of BVDV-2 Genotypes by Real time RT-PCR using ear

notch biopsies during second round of testing

82

14(a)

Detection of BVDV-1 Genotypes by Real time RT-PCR using sera

during first round of testing

83

14(b)

Confirmation of amplified products (BVDV-1amplicons) through

Agarose gel electrophoresis using sera during first round of testing

84

15(a)

Detection of BVDV-1 Genotype by Real time RT-PCR using sera

during second round of testing

85

15(b)

Confirmation of amplified products (BVDV-1amplicons) through

agarose gel electrophoresis using sera during second round of testing

86

16


during first round of testing

87

17


during second round of testing

88

18 (A, B)

Immunohistochemical staining of ear notch biopsies by Alkaline

Phosphatase Naphthol Detection Kit

89

18 (C, D)

Immunohistochemical staining of ear notch biopsies by Alkaline

Phosphatase Naphthol Detection Kit

90

X

19 (A, B)

Immunohistochemical staining of ear notch biopsies by Peroxidase-

DAB Detection Kit

91

20 (A, B) Biotyping of Isolates. 92

CHAPTER 1

1

INTRODUCTION

Bovine viral diarrhoea virus (BVDV) is an important viral pathogen of cattle all over the

world (Gunn et al., 2005; Wegelt et al., 2011) but it can also infect sheep, goats, camels, swine

and even wild species (Nettleton, 1990; Frolich & Streich, 1998; Vilcek & Nettleton, 2006). The

infection caused by this virus was first described by Olafson and co-workers in New York State

as an acute and often fatal disease (Olafson et al., 1946). After few years, Ramsey and Shivers

reported an apparently less contagious disease with low morbidity and higher mortality in

various states of United States that affected the mucous membranes of alimentary and respiratory

tract of cattle. This new disease was named as mucosal disease (Ramsey and Shivers, 1953).

Infections with BVD virus can vary from subclinical to manifestation of clinical signs such as

oral cavity lesions, pyrexia, decreased milk production, diarrhoea, nasal discharge, haemorrhagic

syndrome, death and abortion (Baker, 1995; Thiel et al., 1996).

The BVD virus is a heterogeneous group of viruses belong to genus pestivirus of family

flaviviridae. These are small (40-60nm), enveloped viruses of spherical shapes and classified into

two genotypes, 1 and 2, based on the genetic diversity (Becher et al., 2003). Each naturally

occurring BVD virus strain exits as a cytopathogenic (CP) and noncytopathogenic (NCP)

biotype. Only CP strains are capable to produce cytopathic effects, while NCP strains cause no

cytopathic effects in cells (Dubovi, 1990). Due to genetic variation within the region encoding

non-structural NS2/3 protein, cytopathic viruses arise from NCP viruses (Kummerer et al., 2000;

Becher et al., 2002). Cytopathic biotypes have only been isolated from mucosal disease (MD)

infected animals. Both genotypes of BVD virus have been associated with reproductive failure,

respiratory and enteric diseases in cattle and can cause persistent (PI) or acute transient infection

INTRODUCTION

2

(TI) in animals. Only NCP biotypes can establish persistent infection by avoiding the induction

of a type I interferon response in the fetus and establishing immune tolerance at the time when

fetus differentiate self from non-self (Brownlie et al., 1989; Charleston et al., 2001; Peterhans et

al., 2010). Persistent animals are usually undersized, unthrifty and have high vulnerability to

other diseases, and frequently suffered from mucosal disease (Sandvik, 2005).

The primary reservoir of BVD virus is PI cattle which are one of the main sources of

spreading infection by shedding the virus particles through body excrements like saliva, nasal

discharge, tears, milk, serum, urine, faeces and semen (Brock et al., 1991). Persistent animals

generally showed a high and persistent viremia (Brock et al., 1998; Rae et al., 1987). Under

experimental conditions, these animals have been shown to transmit infection approximately to

60% susceptible animals within 24 hours (Littlejohns, 1985; McGowan et al., 1993). Horizontal

transmission of the virus may be through inhalation or ingestion of virus-containing body fluids

from either PI or TI animals with BVD virus to in contact susceptible cattle (Duffell and

Harkness, 1985). In addition to this, air transmission over short distances has also been reported;

however at greater distances from PI animals, the spread of infection is slow or missing

(Whitmore et al., 1981).

The economic losses due to infection have been noted due to transplacental infection leading

to reproductive failures, still birth, mummification, abortion, persistency, and secondary

infections (Gibbs & Rweyemamu, 1977; Potgieter et al., 1984a; Duffell & Harkness, 1985; Fray

et al., 2000; Straub, 2001; Ackermann & Engels, 2006). The estimation of economic losses is

complex. It depends on the initial immunity of herd, stage of pregnancy of the dam at the time of

INTRODUCTION

3

infection and the pathogenicity of the virus strain. It may vary from a few 100 to several 1000

dollars in individual herd outbreaks (Duffel et al., 1986; Wentink and Dijkhuizen, 1990; Houe,

1999). The estimated losses in individual herd outbreaks with highly virulent strains in cow dairy

herds varied from a few thousand up to $100000 (Houe, 2003; Alves et al., 1996).

Epidemiological surveys revealed a 0.5 to 2% prevalence of BVDV persistency in the cattle

population in different countries of the world (Brock, 2003; Peterhans et al., 2003; Taylor et al.,

1995). In Pakistan, dairy animals are the major source of milk, mutton, wool, hides, bones and

skin. Approximately 40-50 % of income of rural population is based on livestock but

managemental gaps and infectious diseases are main obstacle in the development of livestock

sector. Among the infectious disease, bovine viral diarrhoea is very important in terms of

productivity losses. Unfortunately little attention has been paid until now to investigate this

problem in Pakistan.

In many countries, control programs are being implemented for eradication of this

economically important disease. The success of all these programs depends on the ability to

detect all PI animals at a young age. Undetected PI calves are the main source of the infection

within herds. Precise and economical assays for confirmation of PI cattle with BVDV would be

best, since it would allow prevention of BVDV spreading on farms.

An array of diagnostic techniques are being used in diagnostic laboratories to detect PI cattle

such as virus isolation (VI), reverse transcription-polymerase chain reaction (RT-PCR),

immunohistochemistry (IHC), and antigen-capture enzyme-linked immunosorbent assay (AC-

ELISA) because a single test is considered not ideal in all situations (Brock, 1995; Dubovi,

1996). The detection of BVDV from blood samples by virus isolation may be hindered by the

presence of colostrum-derived maternal antibodies which can neutralize virus in cattle up to 4-6

INTRODUCTION

4

months of age (Brock et al., 1998; Palfi et al., 1993), thus making the assay unreliable (Brock,

1995; Dubovi, 1996). Immunohistochemistry of skin biopsy samples has recently been shown to

be a useful method for screening cattle for persistent BVDV infection, but its reliability is also

questioned due to many reasons (Haines et al., 1992; Njaa et al., 2000; Thur et al., 1996).

In Prince Edward Island Canada, scanty information regarding prevalence of BVDV

persistency, its genotypes and suitability of diagnostic test(s) is available. Due to ever increasing

milk and meat demand, cattle population is increasing in the world and control programs

including accurate identification and culling of persistently infected animals should be initiated.

To achieve this goal, information regarding status of BVD, its genotypes and efficient diagnostic

tests are of utmost importance. Currently serum or ear notches are in use for the detection of

persistent infection with some advantages and disadvantages. Keeping in view the significance

of BVDV persistent infection, the present project has been designed to achieve the following

objectives:

1) Comparison of diagnostic suitability of serum and ear notch biopsy samples using Real time-RT

PCR.

2) Prevalence of BVDV persistency in the defined area.

3) Comparison of various diagnostic approaches for the identification of BVDV persistency, on ear

notch biopsies.

a. Virus isolation

b. Antigen Capture ELISA

c. Immunohistochemistry

d. Real-time RT-PCR.

4) Genotyping and establishing biotypes of detected viruses in the study.

CHAPTER 2

5

REVIEW OF LITERATURE

Bovine viral diarrhoea virus belongs to genus Pestivirus within Flaviviridae family along with

Classical Swine fever virus, Border disease virus (Heinz et al., 2000). Pestiviruses are small (40-

60 nm) enveloped with a non-helical and probably icosahedral nucleocapsid (Fig. 2) (Horzinek,

1981; Francki et al., 1991).

The viral genome is genetically variable. It is non segmented, single stranded positive sense

RNA of about 12.5 kb long. The genome has an untranslated region (UTR) of 360-385 bases at

5' end followed by a large open reading frame with coding capacity for 3898-3988 amino acids

or 435-449 kDa of proteins (Moennig and Plagemann, 1992). The initiation of translation is

mediated by an internal ribosomal entry site. The large open reading frame (ORF) is translated

into a polyprotein that is then cleaved into structural and non-structural proteins by viral and

cellular proteases (Fig. 3). The first synthesized protein responsible for cleavage of its own C

terminus is a non-structural autoprotease (p20), followed by the structural protein such as the

putative core protein (p 14 or C), membrane-associated virion glycoproteins, Erns

(gp48), E 1

(gp25), E2 (gp53) and P7 (Theil et al., 1991; Ruimenapf et al., 1993; Stark et al., 1993). Due to

antigenic and genetic variability, E2 protein is used to study the molecular and serological

diversity of BVD virus. It possesses neutralising epitopes suggesting a significant role in

receptor-mediated viral entry and induction of protective immunity (Bolin et al., 1988; Corapi et

al., 1990; van Zijl et al., 1991; Ridpath et al., 1994; Becher et al., 2003; Pankraz et al., 2005).

Antibodies to Erns

may also be virus-neutralizing (Weiland et al., 1992). The rest of the ORF

encodes non-structural proteins including a putative RNA polymerase and NS 2-3. Both of the


6

proteins are relatively immunodominant of approximately 125 kDa. A smaller part of the NS2, 3

proteins (p80 or NS3) is found in all cytopathic biotypes of BVD virus and the generation of this

protein appears to be associated to the development of mucosal disease (Meyers et al., 1991).

Presently on the basis of non-coding nucleotide sequence at 5’UTR, BVDV isolates have

been divided in two genotypes 1 and 2, which are further divided into subtypes. Each genotype

of virus has two biotypes, CP and NCP (Fig. 1). This biotype division does not correlate with

virulence in the animal but it depends on the potential of the virus to induce cytopathic effects on

cultured cells. The CP biotypes have only been isolated from mucosal disease infected animals

which arise from NCP viruses after genetic variation in non structural protein 3 (Brownlie, 1990

; Meyers & Thiel, 1996 ; Baroth et al., 2000; Kummerer et al., 2000; Becher et al., 2002; Birk et

al., 2008; Neill et al., 2008). The NCP biotype frequently establishes persistent infection in

animals (Kelling, 2004). Noncytopathic strains have a tropism for leucocytes, hair follicles,

lymphoid organs and the respiratory tract, while CP strains are more or less confined to the

digestive tract.

To date, based on the comparison of sequences derived from three genetic regions: 5′-

non-coding region, Npro

and E2, genotype 1 of BVD virus has been classified into 11

subgenotypes (Vilcek et al., 2001) and genotype 2 of BVD virus into two subgenotypes (Flores

et al., 2002; Vilcek et al., 2004). This heterogeneity is due to the fact that during virus

replication, there are chances of high mutation due to the error prone viral RNA polymerase

(Bolin & Grooms, 2004). In spite of, chances of high rate of mutation among BVDV strains, the

viruses isolated from a single herd show high degree of genetic stability after transmission. This

has led to the concept of herd-specific strains of BVD virus (Paton et al., 1995a; Hamers et al.,

1998; Vilcek et al., 1999). Genotype 1 of BVD virus has worldwide prevalence while genotype 2


7

is restricted to USA, North and South America. Some sporadic cases of BVDV2 has also been

reported in Europe and Asia (Beer et al., 2002; Flores et al., 2002; Park et al., 2004; Cranwell et

al., 2005; Barros et al., 2006; Pizarro-Lucero et al., 2006).

Fig. 1: BVDV Classification.

In 1993, severe outbreaks of mucosal disease like, type 2 BVDV infections in cattle herds

have been reported in the USA and Canada, characterized by high body temperature,

thrombocytopenia, pneumonia, haemorrhagic disease, abortions, decreased milk yield ,

sloughing of the mucosa and sudden death (Stoffregen et al., 2000; Alves et al., 1996; Carman et

al., 1998). Forty percent morbidity and twenty percent mortality in affected dairy animals have


8

been noticed. Recently, a third genotype of BVDV “an atypical isolate HoBi”, from pooled

foetal calf serum has also been reported (Schirrmeier et al., 2004).

Fig. 2: Pestivirus Structure

Fig. 3: Genome organization

The virus after getting entry into specific host first replicates in the nasal mucosa, tonsils

and then spreads to the lymph nodes leading to general viraemia (Bruschke et al., 1998). The

initial virus replication may cause mild nasal discharge, stomatitis and erosions in some acute


9

infections (Baker, 1987). After infection, virus can be isolated from nearly all tissues, and

different biotypes can be recovered from different sites whether it is acute infection, mucosal

disease, or a persistent infection. The first step in viral RNA replication is synthesis of minus

strand RNA as template for synthesis of additional plus-strand RNA molecules. Both strands of

viral RNAs can be detected at 4 hours after infection and progeny virus can be detected as early

as 8 hours (Lee et al., 2005).

In 1946, bovine viral diarrhoea (BVD), a new infection in cattle was originally described

by Olafson and colleagues in New York State, USA. The new disease with unknown origin was

associated with epizootics of an acute, fatal, highly contagious disease characterized by fever,

leukopaenia and diarrhoea (Olafson et al., 1946).

In the same year, Childs reported a similar disease named “X disease” in cattle of western

Canada characterized by fever, watery and bloody diarrhoea, dehydration, tachypnea, anorexia,

nasal discharge, hypersalivation and development of ulcers of the mucous membranes (Childs,

1946). At that time, researchers gave it the name, viral diarrhoea (VD), due to most prominent

clinical manifestation-diarrhoea. In 1953, Ramsey & Chivers described a new disease in USA

that affected the mucous membrane of digestive and respiratory tract. The name mucosal disease

(MD) was given to this new disease. This disease appeared to have few similarities to viral

diarrhoea. Finally in 1957, researchers isolated and cultured a virus from a case similar to MD.

The virus was cytopathic (CP) to the cultured cells, causing morphological changes such as

vacuolation and cell death (Underdahl et al., 1957). In the same year, a noncytopathic (NCP)

virus from cases of typical VD of cattle was isolated (Lee & Gillespie, 1957). The relationship

between these 2 isolates was unknown at the time. In 1960, Gillespie and co-workers at Cornell

University also reported a CP virus from a VD infected cattle in Oregon. Thus the name Oregon


10

C24V was given to this isolate. This isolate reproduced the clinical signs that resembled VD and

antibodies that neutralized both CP and NCP strains of VD virus (Gillespie et al., 1960). After

the isolation of virus, the name Bovine Viral Diarrhoea (BVD) was given to VD.

In the late 1960s, a general consensus about BVD of cattle and MD had emerged. Bovine

Virus diarrhea of cattle was considered to be associated with enzootic disease with sporadic

outbreaks with high morbidity but low mortality while MD was observed in young cattle with

low morbidity but high mortality (100%). The similarities in the signs and lesions lead to the

speculation that MD and BVD of cattle were the same disease with minor variations (Jubb &

Kennedy, 1963).

Between late 1960s and 1970, research on pathogenesis of the bovine viral diarrhea-mucosal

disease complex (BVD-MD), particularly in pregnant cattle and neonatal calves was done. From

these experiments, it was revealed that neonatal calves infected with BVDV during gestation

were weak, and that they usually could not survive for more than few months. These calves were

found to be persistently infected (PI), sero-negative to BVDV, and eventually succumbed to MD

(Malmquist, 1968). The diagnosis of NCP BVDV in a sero-negative healthy bull more than 2

year of age further contributed to the eventual elucidation of BVDV persistent infections (Coria

& McClurkin, 1978).

Bovine viral diarrhoea (BVD) disease now has a worldwide distribution. In addition to

cattle the virus also infects sheep, goat, swine and other wild ruminants (Frolich and Streich,

1998).

The prevalence of BVDV infection can be determined by detecting antibody carriers or

persistent animals. The prevalence of antibody carriers animals varies (40% to 90%) in different

countries depending upon the housing system and management (Niskanen et al., 1991; Houe,

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-3XF07M0-3&_user=1069243&_coverDate=08%2F23%2F1999&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=8df86e33c9a80fcb79978944c25ed245#b2http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-3XF07M0-3&_user=1069243&_coverDate=08%2F23%2F1999&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=8df86e33c9a80fcb79978944c25ed245#b1


11

1995 ; Kirkland, 1996), while a prevalence of PI animals ranging from 0.5% to 2% has been

reported (Houe, 1999). In Pakistan, 9 to 12 % prevalence rate of BVDV infections has been

reported on the basis of serology. In United Kingdom, 62.50 % prevalence has been

demonstrated in a serological survey conducted on 1593 cattle (Harkness et al., 1978). In

Denmark and Norway, 64% and 18.50% herds were found positive for antibodies while 1.1%

and 1.04 % were positive for PI, respectively (Houe and Meyling, 1991; Loken et al., 1991;

Cordioli et al., 1996). In United States and Germany, 15% and 45% of the herds were found PI in

a survey of 20 and 329 herds respectively (Houe, 1996; Frey et al., 1996).

The BVDV mainly spreads by contact and through nasal discharge, saliva, semen, urine

and milk of persistent animals (Niskanen et al., 2000; Traven et al., 1991). Acutely infected

animals also secrete virus but the rate of infection spread is low as compared to PI animals

(Brownlie et al., 1987). Indirect routes of transmission like contaminated biologics (vaccines),

fomites, infected semen, contaminated gloves used during artificial insemination, and embryos

transfer from infected animals has also been reported (Schlafer et al., 1990; Kirkland et al., 1991;

Falcone et al., 1999; Barkema et al., 2001; Givens et al., 2003; Niskanen & Lindberg, 2003;

Schirrmeier et al., 2004; Stringfellow et al., 2005).

Different clinical forms of BVD infection have been described (Tremblay, 1996).

Initially the reported spectrum of clinical signs in postnatally infected sero-negative cattle was

limited subclinical to mild infection, followed by negligible mortality at any age, production of

neutralizing antibodies and rapid recovery (Duffel and Harkness, 1985). The clinical aspects of

the infection of pregnant animals depend on multiple interactive factors including immune status

to BVDV, the gestation period and type of the virus, however in non-pregnant cattle; infection is


12

usually mild and is often overlooked by the farmers (Baker, 1995; Evermann & Barrington,

2005).

Acutely infected animals show unavoidable pyrexia, a mild nasal discharge and

leukopenia for 3 to 7 days post infection. For a period of about 3 to 14 days post infection, virus

can be isolated from the blood and nasal secretions. The antibodies titers rise to maximum level

10 to 12 weeks after infection. Immunity is supposed to be lifelong (Duffell and Harkness, 1985;

Fredriksen et al., 1999). In immunocompetent animals, infection leads to transient

immunosupression which in turn could aggravate other diseases such as bacterial

bronchopneumonias, coronavirus, parainfluenza virus 3, infections and mastitis (Potgieter, 1995;

Liebler-Tenorio, 2005; Berends et al., 2008).

It is usually accepted that the economic losses of BVD virus infections are in terms of

reproductive dysfunction, reduced milk production, poor growth rate, high culling rate, and

treatment expenses (Baker, 1995; Houe, 1995; Houe, 2003; Waage, 2000). The main economic

impact of BVDV infections, however, is caused by infections in the seronegative pregnant

animals that result in transplacental transmission and foetal infections (Grooms, 2004).

Depending on the gestational age of the early conceptus or foetus, a wide range of reproductive

disorders like embryonic deaths, abortions, malformations, birth of stillborn or weak calves, or

birth of PI calves can be seen in infected animals. Abortions are most common during the first

trimester but may occur at any time during pregnancy. Foetuses infected from around 30 days in

gestation, and until the foetus becomes immunocompetent at around 120-125 days in gestation,

may be born persistently infected (PI) with BVDV. PI calves become immunotolerant to the

infecting strain, and remain sero-negative. If exposed to a heterologous strain, however, they

may develop low level of antibody (Bruschke et al., 1998; Fulton, et al., 2003b). They are often

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-4H8FPJH-4&_user=1069243&_coverDate=11%2F15%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_searchStrId=962794549&_rerunOrigin=scholar.google&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=c535a59ced55c1410d613863cde4e586#bib2http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-4H8FPJH-4&_user=1069243&_coverDate=11%2F15%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_searchStrId=962794549&_rerunOrigin=scholar.google&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=c535a59ced55c1410d613863cde4e586#bib30http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-4H8FPJH-4&_user=1069243&_coverDate=11%2F15%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_searchStrId=962794549&_rerunOrigin=scholar.google&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=c535a59ced55c1410d613863cde4e586#bib32http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBK-4H8FPJH-4&_user=1069243&_coverDate=11%2F15%2F2005&_rdoc=1&_fmt=full&_orig=search&_cdi=5145&_sort=d&_docanchor=&view=c&_searchStrId=962794549&_rerunOrigin=scholar.google&_acct=C000051268&_version=1&_urlVersion=0&_userid=1069243&md5=c535a59ced55c1410d613863cde4e586#bib66


13

born weak and undersized, but many appear normal at birth. If developing fetus in dam gets

infection after 150 of gestation, the immune system of the fetus will be competent enough to

develop specific immunity leading to the birth of a normal calf.

Due to an impaired immune system of PI animals, they are particularly susceptible to

other infections, which partly explain the high mortality during their young age, compared to

non-infected calves (Houe, 1999). Some PI animals remain clinically unaffected and may breed

satisfactorily leading to birth of PI progeny (McClurkin et al., 1979). Foetuses infected during

the later stages of gestation develop immune response competent enough to clear the virus.

Despite this, many congenitally infected animals experience serious postnatal health effects

(Munoz-Zanzi et al., 2003).

When a PI animal is co-infected with a CP biotype of the virus, it always leads to a fatal

mucosal disease (MD). The source of the super infecting CP biotype is either endogenous, i.e.

due to an alteration in the genome of NCP biotype within the PI animal, or exogenous due to

presence of MD infected animals in proximity or from a live BVDV vaccine (Brownlie, 1990).

Mucosal disease is always fatal with rapid onset. The appearance of dead or moribund animals is

first sign seen in the herds. The infected animals show abdominal pain, become anorexic,

reluctant to move and may develop a profuse diarrhea along with excessive salivation and

lacrimation. Necropsy examination reveals erosions at various sites in gastrointestinal tract

particularly along the gingival margin particularly in the lymphoid Peyer’s patches in the small

intestine and in the ileocaecal lymph nodes.


14

CONTROL OF BVDV

To control BVDV infection, different control strategies, either with or without vaccination are

being used in many countries including Denmark, Finland, Sweden, Norway and Austria, with

success (Rossmanith et al., 2005; Valle et al., 2005; Hult & Lindberg, 2005; Rikula et al., 2005).

For a long time, control measures were limited, only to prophylactic vaccination practices to

reduce or prevent clinical disease in the herds. The primary objective of vaccination was to

induce enough immunity which can prevent infection of foetuses in-uterus and persistency.

However, recognition and culling of PI animals is obligatory for the eradication of BVDV from

the herds (Lindberg & Alenius, 1999; Brock, 2004b).

Consequently, modern vaccination programmes are designed not only to prevent clinical disease,

but also to prevent viremia and foetal infection (Kelling, 2004). The results of these vaccination

programs are controversal. Under controlled experimental conditions inactivated as well as live

vaccines may prevent foetal infection (Cortese et al., 1998; Frey et al., 2002; Patel et al., 2002),

but under field conditions, the efficacy of these vaccines has been questioned (van Oirschot et

al., 1999; O'Rourke, 2002; Graham et al., 2003). Births of PI calves in vaccinated herds have

been reported (Van Campen et al., 2000; Gaede et al., 2004; Graham et al., 2004). The antigenic

diversity among BVDV field strains along with various other factors, like inconsistent use of the

vaccines and biosecurity breaches, may lead to vaccine failures. It is well accepted that to

prevent infection after eradication, 100% efficacy of vaccines is needed. Once the infection is

introduced, widespread use of vaccination failed to reduce the occurrence of BVDV (Lindberg &

Houe, 2005).


15

In the last decade, a non vaccination strategy to control BVDV has been initiated in

Europe, and particularly in the Scandinavian countries. It was based on an initial determination

of BVD virus status in the herd, identification, culling and preventing re-introduction of PI

animals in non-infected herds (Lindberg & Alenius, 1999; Greiser-Wilke et al., 2003; Sandvik,

2004). These programmes proved to be successful, and all of the Scandinavian countries are

currently either free, or almost free of BVD virus.

Based on the success of the control programmes in Scandinavia, it is now well established that

all three elements: i.e, biosecurity, virus elimination and monitoring should be included in the

control program.

DIAGNOSIS

To achieve successful prevention and control of an infectious disease, there must be adequate

methods for the diagnostic detection and identification of the pathogen in a timely manner.

Various diagnostic assays aimed to detect virus specific antibodies and infectious virus/viral

component are available to determine the status of BVD virus in the herds (Sandvik, 2005). The

main objectives of diagnostic assays are to discriminate infected from non infected herds, to

monitor success of control programme and identification of persistent animals (Lindberg &

Alenius, 1999). Different diagnostic methods used are the following:

SEROLOGICAL METHODS

Serological methods can also be used to diagnose acute infection by detection of humoral

immune response with follow up re-sampling. For the detection of sero-conversion, various


16

serological assays have been used for BVD virus. Among these, serum neutralisation and

enzyme linked immunosorbent assays (ELISA) are considered more sensitive.

SERUM NEUTRALIZATION TEST (SNT)

Serum Neutralization (SN) tests is taken as gold standard for antibody titration. It is specific and

sensitive, but due to involvement of cell culture is labour demanding and will take 5-6 days to

perform. Thus, it is usually used as a for back-up test for reference (Sandvik, 2005). The

antibodies detected are mainly against E2 protein of virus and antibody titre in the same sample

may vary depending upon the strain of virus used in the assay (Jones et al., 2001; Couvreur et al.,

2002). Cytopathogenic strains (Oregon C24V and NADL) of BVD virus are usually used for

titration of antibodies. Now immune conjugates based assays are available that permit detection

of neutralizing antibodies against non-cytopathic biotype of viruses. Pooled samples for

determination of antibodies level against BVD virus can give indication about the status of

BVDV in a herd (Niskanen, et al., 1991; Niskanen, 1993; Houe et al., 1995; Paton et al., 1998;

Lindberg & Alenius, 1999; Pritchard, 2001; Valle et al., 2005). Bock et al., (1997) determined

the proportion and incidence of PI calves with pestivirus in Australian herds. Serum

neutralization (SN) and an antigen-capture ELISA (AC-ELISA) tests were applied to determine

antibody and antigen to bovine pestivirus respectively. The calves were also examined for

pestivirus by inoculating pooled lymphocyte samples from calves in the sheep. The study

included eight herds. Serum neutralization test was used as screening test and antigen-capture

ELISA as follow up test. The animals having SN antibody titers


17

for confirmation of pestivirus antigen. Out of total 1521 animals, 0.9% (14) was found PI with an

incidence ranging from 0.0 to 3.0 % per year over 6 years. In the study, off eight test herds, 04

were found with PI animals. Based on the findings, it could be concluded that sheep inoculation,

paired AC-ELISA and SN tests in combination can be used for detecting persistently infected

calves with bovine pestivirus with highly sensitivity and specificity. In another study, virus

neutralization test was used to measure the neutralizing antibodies to genotype 1 and 2 of bovine

viral diarrhea virus using cell culture. The presence of antibodies can be confirmed by inhibition

of viral cytopathology or by immunoperoxidase staining for cytopathic and noncytopathic strains

respectively. Monoclonal antibody 15C5 specific for BVD virus, biotinylated rabbit anti-mouse

antibody, horse reddish peroxidase-streptavidin and 3-amino-9-ethyl carbazole as substrate was

used. Twenty strains of BVDV consisting of 14 of type 1 and 6 of type 2 were used to infect

cells in the lab. The serum containing antibodies against both type 1 and 2 was used as positive

control serum. Regardless of biotype, no significant differences in antibody titers for respective

type strains, was observed. It was also found that calves vaccinated with either modified live

virus or inactivated vaccine (BVDV type 1) depicted higher antibody response to type 1 strain

compared to type 2 strains. Thus, although, the genotypes are differentiated by non-coding

sequences, there appears to be more vigorous virus neutralizing Abs response by genotype

homologous antibody (Fulton et al., 1997).

ANTIBODY CAPTURE ELISA

The ELISA test is advantageous by SNT for being rapid, relatively inexpensive, and easy to

establish and run. Large number of samples can be processed within short time. Two different

ELISA formats are in use to determine the antibody status of the herd: indirect or blocking


18

(competitive) assays. In the indirect format, the ELISA plates are coated with viral antigen and

specific antibodies are trapped by immobilized viral antigen. The specific reaction is

subsequently detected using enzyme conjugated species-specific anti-antibodies. A positive

reaction is interpreted reading the optical density (OD) of color which developed on addition of

substrate solution. In blocking ELISAs, conjugated virus-specific antibodies binding to adsorbed

antigen is blocked by virus-specific antibodies in the sample. Thus the positive sample will

express no or low OD relative to negative reference serum.

DETECTION OF BVDV

In principle, three classes of methods like detection of virus, its nucleic acid and virus isolation,

are in use. Blood, serum, faces and skin biopsies of infected animals can be used for detection of

BVD virus and viral genome (Sandvik et al., 1997a; Bruschke et al., 1998; Ellis et al., 1998).

From persistently infected animals, BVDV antigen can be detected throughout their life.

Commonly used methods include virus isolation, different immune based antigen detection

assays, such as ELISA or immunohistochemistry (IHC), and reverse transcriptase-polymerase

chain reaction (RT-PCR). Virus isolation and AC-ELISA, however may be negatively influenced

by maternal antibody, while, IHC and PCR have proved to be effective even in the presence of

antibodies (Zimmer et al., 2004; Kuhne et al., 2005; Njaa et al., 2000; Horner et al., 1995).

VIRUS ISOLATION (VI)

BVDV was first isolated as a cytopathogenic agent in bovine kidneys cell cultures (Underdahl et

al., 1957). BVD virus has been isolated in numerous types of bovine cell cultures such as bovine

fetal kidney, bovine turbinate cells, bovine testicular cells, Madin Darby Bovine Kidney


19

(MDBK) and bovine endothelial cells (Sandvik, 2005; Cornish et al., 2005). BVD virus is

relatively easy to isolate in cell cultures. CP strains of BVDV induce cytopathic changes on

cultured cells within 48 hours post inoculation. However, generally, BVD field virus isolates are

non-cytopathic. Virus isolation using bovine cell cultures, followed by confirmation through

immunoperoxidase or immunofluorescence staining is virus isolation (VI) in bovine cell cultures,

is considered to be the standard test (Meyling, 1984). For confirmation of NCP strains, usually 3

to 5 days are required. Serum, blood, nasal swabs, semen and tissues samples may be used for

diagnosis of BVD virus. White blood cells are most commonly used for screening of neonatal

calves but use of VI test in neonatal calves is not dependable due to the presence of passively

derived maternal antibodies, or cytotoxic sera, both of which can yield false negative results

(Bolin et al., 1991). Moreover, it is compulsory that fresh cell cultures must tested before use to

rule out any viral contaminants (Bolin et al., 1994; Edwards, 1993). Liquid nitrogen can be used

to preserve primary or secondary cultures in frozen form. Bovine viral diarrhea virus free cell

lines can be maintained by the use of continuous cell line through regular testing (Bolin et al.,

1994). The fetal bovine serum used to supplement the cell culture should be free from both

BVDV and its neutralizing antibody (Edwards, 1993). Irradiation of BVDV in serum at 25

KiloGrays (2.5 Mrad) is more reliable to inactivate the virus than that of heat treatment at 56°C

for 30–45 minutes. However, irradiated commercial batches of fetal bovine serum remained

positive by PCR. Bovine fetal serum may be replaced by horse serum. Buffy coat, whole blood,

leukocytes or serum are suitable for isolation of the virus. Maternal antibodies may interfere

isolation of BVD virus from the serum samples. Therefore procedures for virus isolation should

be optimized to give maximum sensitivity.


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ANTIGEN CAPTURE ELISA (AC-ELISA)

Several formats of ELISA are commercially available for detection of viral antigens. The AC-

ELISA is mostly based on MAb specific to viral antigens (Fenton et al., 1991; Mignon et al.,

1992; Shannon et al., 1993; Shannon et al., 1991). The basic principle is based on the use of

virus-specific monoclonal antibodies reaction with capture viral antigens and its detection by

enzyme-conjugated antibodies. Antigen capture ELISA is widely used for identification of PI

animals, and can be used for detection of virus in serum, buffy coat cells or skin biopsies (e.g.

ear notch samples). Antigen capture ELISA may yield false negative results if antibodies are

present in the sample. This should be considered when testing blood based diagnosis in young

animals that might have persisting maternal antibodies (Zimmer et al., 2004). In a study

conducted by Mignon et al. (1992), Bovine viral diarrhea virus was detected in blood samples by

an enzyme-linked immunosorbent assay (ELISA). A total of 761 samples of known status

(viraemic or not) were evaluated. The sensitivity, specificity and predictive values of the assay

were 100% compared to that of virus isolation (90%). ELISA was proven good replacement of

virus isolation techniques for detection of BVD virus in persistent animals. In another study,

antigen-capture ELISA (AC-ELISA) was used to detect pestivirus in persistently infected cattle.

Various samples like blood clots, blood leukocytes and tissue samples were tested in this study.

A complete agreement was found between ELISA and conventional virus isolation procedures.

Three broadly-reactive monoclonal antibodies were used to detect captured antigen. Higher

optical densities for blood clots and blood leukocytes from infected animals were observed than

uninfected animals. Spleen and liver samples of carrier cattle had OD values of 1.77 and 0.95

respectively with < 0.20 for negative tissue samples. The AC-ELISA was found to be suitable for

regular diagnostic and certification testing (Shannon et al., 1991). Fulton et al. (2006) evaluated


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the efficacy of vaccine by challenge study using noncytopathogenic BVDV2a. Various tests

were also compared to discriminate BVDV transiently infected calves from PI calves. Ear

notches were collected from persistent and transiently infected animals. Fresh notches were

tested through an antigen-capture enzyme-linked immunosorbent assay and formalinized by

immunohistochemistry test to detect BVDV antigen. Both assays failed to discriminate persistent

animals from transiently infected animals. In another study, for the detection of BVD virus, 860

blood samples without antibodies were tested through both virus isolation and in an antigen-

capture enzyme linked immunosorbent assay (ELISA) based on monoclonal antibodies (MAbs)

against the nonstructural BVD virus protein p125/p80. A total of 843 samples (98%) were

positive (n= 170, 20%) or negative (n = 673, 78%) in both tests, corresponding to an agreement

of K = 0.94. Among 17 samples with diverging results, 3 were from animals transiently infected

with BVD virus, and 5 came from clinically affected animals. The reactivity of the MAbs was

controlled against 387 field isolates of BVD virus. All were detected by the MAbs, thereby

confirming the general view that the p125 virus protein is highly conserved among different

BVD viruses (Sandvik and Krogsrud, 1995). Kuhne and colleagues applied an antigen capture

enzyme linked immunosorbent assay on ear notch biopsies from cattle to detect bovine viral

diarrhoea virus (BVDV). After processing a total of 99 BVDV positive and 469 negative

samples, a sensitivity of 100% and specificity of 99.6% was found. It was also found that after

intake of colostrums, positive serum samples turned negative while ear notch biopsies remained

positive all the times for BVDV. Testing multiple ear samples from PI cattle yielded consistently

positive results. The author concluded that, ear samples testing through ELISA could be used as

a reliable and economic way of BVDV testing (Kuhne et al., 2005). Efficacy of 2 commercial

antigen capture enzyme linked immunosorbent assays to detect bovine viral diarrhoea virus


22

(BVDV) in serum and skin biopsies was evaluated by Hill et al. (2007). Ear notch biopsies and

serum samples were collected from 30 known persistently infected cattle and 246 cohorts as

well. Skin biopsies elutes were collected after soaking overnight in buffer. Both elute and sera

were tested through two commercially available ELISAs for detection of BVDV antigen.

Furthermore, to validate the results of ELISAs, a subsample of positive and negative sera was

also tested using a polymerase chain reaction (PCR) test. A study was also undertaken to

determine the possibility of cross contamination that may occur during collection and processing

of skin tissues. All the samples which were found positive for persistent infection through either

ELISA remained positive by PCR showing a perfect agreement between all assays. No evidence

of cross-contamination during collection and processing of skin samples was observed in this

study.

IMMUNOHISTOCHEMICAL ASSAYS

In the recent years, a new technique “immunohistochemistry (IHC)” for the detection of BVD

virus using skin biopsies had been introduced earlier by Thur et al. (1996).

Njaa et al. (2000) detected positive staining in 41 of 42 formalin-fixed, paraffin-embedded skin

samples from persistently infected calves using peroxidase based IHC technique. The ear skin

biopsy is now being used to screen herds for persistently infected cattle particularly for screening

of young calves due to relative ease in collection of sample and independence from risk of

interference with persistent maternal antibodies (Brodersen, 2004). Driskell and Ridpath, (2006)

assessed current BVDV detection methods being used at various laboratories in USA. Data from

26 veterinary diagnostic laboratories in 23 states was collected which revealed no clear

consensus on BVDV testing method. Further, it is found that that ear-notch antigen capture

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Hill%20FI%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus


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enzyme-linked immunosorbent assay (ACE) was the test most commonly used test for the

detection of BVDV. Groom and Keilen, 2002 evaluated the use of peroxidase based

immunohistochemical staining (IP-IHC) for early detection of persistent BVDV infection using

skin biopsy samples from neonatal calves. A total of 332, 1 to 4-week-old dairy calves were

screened for BVDV. Immunohistochemistry (IHC) staining results for BVDV antigen on

formalin-fixed skin biopsy samples were compared to those of virus isolation (VI) from white

blood cell preparations. Six calves were taken as persistently infected with BVDV by both IHC

and VI tests. Virus isolation detected one acutely infected calf which was found negative by

IHC. However, on follow up test, the calf was tested negative by VI. Thus,

immunohistochemical staining of skin biopsy samples was found a reliable and useful

management tool recommended as in aid of controlling and preventing BVDV infection. Cornish

et al. (2005) compared immunohistochemistry (AP-IHC) and antigen-capture ELISA (Ag

ELISA) on ear notches, for detection of BVDV persistent infection (PI) in 559 Angus calves

aging from 1 and 5 months. Virus isolation and reverse transcription (RT–PCR) tests on buffy

coat for detection of BVDV infection were also applied. Serum neutralization (SN) test was used

to determine level of antibodies to BVDV types 1a and 2. A total of 67 out of 559 (12.0%) calves

tested positive at initial screening by IHC using alkaline phosphatase system, Ag ELISA, or VI

tests. All positive calves were kept for a minimum of 3 months for repeat testing monthly by

IHC, Ag ELISA, VI, RT-PCR, and SN. Of these calves which were positive at initial screening,

59/67 (88.1%) were found PI and 8/67 (11.9%) acutely infected. Both IHC and Ag ELISA

detected 100% of PI calves. In the study however, IHC and Ag ELISA also detected 6 and 8

acutely infected calves, respectively, at initial screening. Furthermore, IHC and Ag ELISA

continued to detect acutely infected calves 3 months after initial screening. Indistinguishable


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IHC staining signals from PI calves, in 3 acutely infected calves were observed at initial

screening. It is recommended that, both IHC (IP-IHC) and Ag ELISA were accurate in detecting

PI animals but both tests also detect some calves acutely infected with BVDV due to which,

repeat testing using VI or RT-PCR on buffy coat samples was suggested, usually at 30 days after

initial screening to conclusively distinguish between acute and PI. Luzzago et al. (2006)

evaluated the reliability and feasibility of IHC using immunoperoxidase label (IP) on ear skin

tissues to detect PI animals in field conditions, including both adult and calves less than 6

months of age. In animals over 6 months of age, skin biopsy and blood sample were collected at

the same time, whereas in young calves blood sampling was performed when animals reached 6

months of age. One hundred and sixty-five animals were tested, and immunohistochemical

results were compared with those of antigen ELISA. In case of inconclusive results, virus

isolation and virus neutralization assays were performed. Agreement K value was 0, 96.

Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of

the epidermis and adnexa. The author concluded that, IP-IHC on skin biopsies is a reliable test

for identification of PI animals, and provides an alternative and/or complementary method to VI

and antigen ELISA, particularly in neonatal calves, where the sensitivity of the latter tests can be

hampered by the presence of maternal antibodies. In addition fixed tissues did not present the

inconvenience of laboratory virus contamination. Provided that prolonged fixation was avoided,

IHC was an inexpensive, sensitive, specific and reliable diagnostic test to identify persistently

infected cattle. Baszler et al. (1995) processed 50 formalin-fixed paraffin-embedded tissues from

spontaneous cases (39 bovine, nine ovine, two caprine) of bovine viral diarrhea virus (BVDV)

infection by virus isolation and alkaline phosphates based immunohistochemistry (IHC) using

anti-BVDV gp-43 monoclonal antibody (Mab 15C5). In the study, virus isolation and IHC was


25

compared in determining BVDV

and cellular distribution of BVDV in various

clinical

manifestations of infection. In bovid with abortion enteric (mucosal disease, acute and chronic

diarrhea, neonatal diarrhea) and respiratory disease, 100% concordance

of virus isolation and

immunohistochemistry was found. When laboratory tests applied on gastrointestinal tissue

and/or feces, immunohistochemistry detected

100% BVDV cases whereas, virus isolation

detected BVDV in only 65% of cattle. In all clinical forms of BVDV infection, distribution of

BVD virus was widespread in various tissues of individual cattle. In the absence

of other

pathogens, viral antigen accumulation was correlated with tissue only in the lung, placenta

gastrointestinal tract, lymphoid tissue and eye. This study demonstrated the usefulness of

immunohistochemistry to diagnose BVDV infections in cattle. Hilbe et al. (2007) compared five

diagnostic tests (peroxidase based immunohistochemistry (IP-IHC), 2 commercial antigen

ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR) for the detection of bovine viral

diarrhea virus infection using skin biopsies (shoulder region) and/or serum. A total of 224 calves

(0-3 months of age), 23 calves (>3 months but < 7 months) and 11 cattle (> 7 months) were

included in the study. Both skin and serum samples were found equally appropriate by 3 antigen

detection methods and the real-time RT-PCR. Off 249 samples, 26 were BVDV-positive with all

antigen detection methods and the real-time RT-PCR while 9 out of 258 samples with discordant

results were retested by RT-PCR, RT-PCR reamplification (ReA), and antigen ELISA I on

serum. Immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies was also

performed. These discordant samples were also processed for virus isolation and subsequently

for genotyping. Transiently infected animals were identified in 3 cases while 2 samples which

were tested positive by real-time RT-PCR were recognized false positive due to cross-

contamination. Due to the presence of maternal antibodies, the antigen ELISA II failed to detect


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2 BVDV-positive calves. The cause of false-positive results in this ELISA remained uncertain.

The author concluded that, only IHC (IP) or antigen ELISA I assays on skin samples can be

efficiently used to detect persistently infected animals. Thur et al. (1997) demonstrated BVDV in

fetuses by peroxidase-immunohistochemical (IP-IHC) methods on cryostat and paraffin sections,

by virus isolation in cell culture and in some instances, an antigen capture ELISA.

Immunohistochemical methods and virus isolation in cell culture sensitivity for detection of

BVD virus was equal; nevertheless, it decreased during autolysis. In such cases, use of paraffin-

embedded, formalin-fixed brain sections was the most suitable method whereas; antigen

detection by ELISA was less sensitive. In this study, it is concluded that immunohistochemical

analysis of cryostat sections of thyroid gland, brain, skin, placenta and abomasum, is a fast,

sensitive method for detecting pestiviruses in fetuses. Formalin-fixed, paraffin-embedded brain

sections were mostly recommended among other described methods in the presence of advanced

autolytic changes.

POLYMERASE CHAIN REACTION (PCR)

Reverse transcription-polymerase chain reaction (RT-PCR) is a quick and sensitive technique for

detection of viral RNA. In the conventional PCR protocols, various steps (extraction of RNA,

reverse transcription to cDNA, amplification and detection of amplicons) are carried out

separately, which is time-consuming. The necessity of opening the PCR tube for product

detection increases the risk of false positive results due to amplicon contamination.

More recent real-time RT-PCR systems minimize these drawbacks, as after RNA extraction, all

steps are carried out in a single tube thus eliminating the risk of carry-over contamination

(McGoldrick et al., 1999). The Real time PCR assays are excellent tools for rapid identification

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of viral nucleic acids, mutation analysis, genotyping of various field isolates, studying viral load

and epidemiology (Ginzinger, 2002; Mackay et al., 2002). Simultaneous quantification, detection

and genotypes of causative agents can be accomplished by the use specific primers and probes in

the same assay (Letellier & Kerkhofs, 2003). In these assays, the quantification is done by

determining the cycle threshold (Ct value) through real time fluorescence monitoring during the

exponential growth phase of PCR reactions (Mackay et al., 2002; Ong and Irvine, 2002). Ct

value is taken as the PCR cycle at which the product specific fluorescent signal is significantly

higher than the average background signal. It is actually the point at which PCR amplification

enters the exponential phase. Various chemistries to generate the fluorescent signals are being

used. These chemistries can be, sequence independent or sequence specific. The sequence

independent dyes as SYBER Green1, YOPRO-1, ethidium bromide, Thiazole orange, yellow

orange, and Enhan CE bind to ds DNA molecules and emit fluorescence upon excitation and do

not bind with ss DNA. (Garcia-Canas et al., 2002; Ginzinger, 2002; Mackay et al., 2002) Among

these dyes, SYBER Green1 is perhaps the most widely used. It is a minor groove binding dye

(Bustin, 2000; Mackay et al., 2002). The major disadvantage is its non-specific binding to any

double-stranded DNA, including primer dimers and non-specific products, so specificity is

determined only by specific primers (Bustin, 2000; Skeidsvoll and Ueland, 1995). A melting

curve analysis is needed to be performed at the end of the reaction to differentiate specific

signals from non-specific signals (Bustin, 2000; Mackay et al., 2002). In contrast to SYBER

Green 1, sequence specific chemistry is based on the ability of confirmatory probes(s) with the

fluorescent label(s) to bind its complementary sequence on one or both strands of the target

DNA. These formats include TaqMan (Hydrolysis) probes, displaceable beacons, cleavable

beacons and Amplifluor Uniprimer system (Bustin, 2000; Ginzinger, 2002; Mackay et al., 2002).


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TaqMan chemistry is based on the ability of 5’ to 3’ nuclease activity of Taq or Tth DNA

polymerase to generate a fluorescent signal by the cleavage of fluorescent reporter at the 5’ end

of the probe when it hybridized to its complementary sequence (Bustin, 2000; Mackay et al.,

2002). TaqMan probes are also called hydrolysis probe because of the fact that they are

hydrolyzed by the nuclease activity of the enzyme. Currently the most popular real-time PCR

assay principle is based on the binding of a dual-labeled probe to the PCR amplicon and the

release of a signal by loss of fluorescence quenching as chain reaction degrades the probe. The

dual-labeled probes used in real time PCR are designed in such a way that they have 5 to 10 C

higher melting temperature (Tm) than the two primers. This allows the probe to remain bound to

its target strand during the primer extension (Bustin, 2000; Ginzinger, 2002). In the recent years,

there has been an increasing interest in the use of real time PCR for detection of BVDV and

other important viruses. Horner et al. (1995) evaluated the suitability of three different tests for

the confirmation of ruminant pestivirus infections. Reference strains of bovine viral diarrhoea

virus (BVDV) and buffy coat samples from persistently infected (PI) carriers were used for

sensitivity studies. Reverse transcription- polymerase chain reaction (RT-PCR) was found with

greater sensitivity than the other tests. Furthermore, the antigen capture enzyme-linked

immnunosorbent assay (ELISA) due to least sensitivity could only be used on tissue or blood

samples. In the study conducted on clinical samples, the RT-PCR detected the most positives

(42/169) compared to the ELISA (32) and the immunoperoxidase test (IPT) (20). The RT-PCR

was found successful even in the presence of specific antibody in the sample. The poor

sensitivity of the IPT was related to testing of toxic or contaminated or the use of a 1 passage (4-

day) test and the samples. For large scale testing for diagnosis and control of pestivirus

infections, ELISA was found to be most suitable assay to be used. Bhudevi and Weinstock


29

(2003) identified BVD virus in freshly processed formalin-fixed paraffin embedded tissue

sections and archival samples from both acutely and persistently infected animals up to 7 years

old by real time quantitative RT-PCR using TaqMan probes. To see the effect of RNA

degradation due to tissue processing and handling, fresh tissue biopsies from a BVDV infected

persistent calves were stored at 4°C or room temperature for up to 7 days before formalin

fixation for 24 hours and histologic processing. Samples which were stored at 4°C for 7 days

prior to fixation were positive while samples kept at room temperature remained positive at 74

hours but turned negative after 96 hours. Mild decrease in signal strength was observed in fresh

tissue fixed in formalin for 1 week prior to processing compared with tissue fixed for 24-48

hours. Real time RT-PCR improved diagnosis of BVD infection by allowing prospective and

retrospective identification of BVD virus in tissues. Kennedy et al. (2006) conducted a study to

detect BVDV persistently infected (PI) animals using ear notch samples. Peroxidase based

immunohistochemistry (IP-IHC), reverse transcription-polymerase chain reaction (RT-PCR) and

individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA) on pooled

supernatants of ear-notch were compared with samples from 3,016 heifers. Individual AC-

ELISA tests were compared with RT-PCR ear-notch pools with samples from all 3,599 heifers.

Only four heifers were tested positive by both AC-ELISA and IHC. When RT-PCR was applied

on each of randomly pooled ear notch supernatant from 100 animals, 2 pools were identified that

contained one positive AC-ELISA sample and 1 pool that contained two positive AC-ELISA

samples. Furthermore, pooled RT-PCR ear notch supernatant detected 100% (n 5 36) samples

which were spiked with supernatant from selected positive AC-ELISA ear notch. Though repeat

confirmatory tests were not completed, all 3 methods showed perfect agreement (100%) in

detecting suspected PI animals (kappa value of 1). The application of RT-PCR on pooled ear-


30

notch supernatant could be a good choice which is rapid, cost-effective for initial screening of

cattle herds for BVDV PI animals. Subsequent testing of individual samples in positive pool by

an AC-ELISA could minimize the risk of virus exposure to other animals due to rapid test

results. Ridpath and Bolin (1998) used polymerase chain reaction (PCR) for classifying BVDV

isolates into genotypes and subgenotypes, CSVF, BDV, BVDV1a, BVDV1b and BVDV2 on the

basis of 5’ un-translated region sequences. A total of 345 previously classified viral isolates from

cattle and small ruminants were used to validate differential PCR tests. A perfect agreement

(100%) was found between classification by differential PCR and the previous segregation of

these viral isolates. Ridpath et al. (2002) studied the ability of polymerase chain reaction

amplification followed by probe hybridization (RT-PCR/probe) of serum samples to detect PI

animals and peroxidase-immunohistochemical for viral antigen in skin biopsies (IHC) to detect

acute BVDV infections. A total of 16 BVD virus and antibody free, colostrum- calves were

challenged with 6 different BVDV strains. Virus was detected 19% acutely infected animals by

the RT-PCR/probe technique while no acutely infected animals were tested positive by IHC.

Mahlum et al. (2002) stated that polymerase chain reaction (RT-PCR) is fast and more sensitive

compared to cell culture isolation; however test results can be compromised by sample

contamination during nucleic acid amplification. In this study a closed-tube format of BVDV

nucleic acid amplification and detection by TaqMan RT-PCR was used and results were

compared with those of virus isolation, IPMA, and IP-IHC. TaqMan RT-PCR detected BVDV in

many samples which were tested negative by IPMA, IHC, and virus isolation. Only one sample

was found was positive by IHC. The study revealed that TaqMan RT-PCR in a closed-tube is a

rapid, economical and sensitive method to be used for BVDV detection without concerns of

amplified cDNA product contamination. Baxi et al. (2006) detected and classified bovine viral


31

diarrhea viruses (BVDV) by one-step multiplex real-time reverse transcriptase-polymerase chain

reaction (RT-PCR) using SmartCycler technology and TaqMan probes. Common primers and

type specific TaqMan probes for genotype 1 and 2 of BVDV were designed in the 5’-

untranslated region of the viral genome. The detection limit of real-time assay was found to be

10–100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1

and BVDV2, respectively. The probes were found highly specific, no reactivity with the closely

related pestiviruses, classical swine fever virus and border disease virus was observed. The assay

accurately classified 54 BVDV strains and field isolates with high reproducibility. There was a

full agreement between one-step real-time RT-PCR assay and virus isolation for bovine serum

samples. One-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for

detection and typing of BVDV. Drew et al. (1999) used a single step, single-tube reverse

transcriptase-polymerase chain reaction (RT-PCR) to detect bovine viral diarrhoea virus

(BVDV) in somatic cells from bulk milk samples. Samples from 80 herds with a history of

BVDV were tested to validate the assay and the findings were compared with those of samples

originating from same sized control group. A total of 20.5% of herds with a history of BVDV

were found positive while all were found negative in control group. The assay proved specific

and sensitive. It detected one persistently infected (PI) animal out of 162 lactating animal herd.

On follow-up blood testing from 19 herds by RT-PCR, ten herds were positive containing at least

one lactating PI animal. The authors concluded that for control strategy aiming detection and

culling of PI lactating cattle at the time of sampling, the test provides a rapid and inexpensive

alternative to individual animal testing for cows.


32

GENOTYPING

The genetic typing of BVDV has most frequently been based on sequence analysis of the 5’

NCR, Npro or E2 regions (Vilcek, et al., 2001; Becher, et al., 2003; Nagai et al., 2004; Toplak et

al., 2004). Analysis of the 5’ NCR, a highly conserved region of the genome, has shown to be a

reliable and reproducible method for genetic characterization of BVDV isolates (Ridpath,

2005b). Furthermore, it is the target region for most PCR-based diagnostics, and as such a

suitable target for direct sequencing from the PCR product. In spite of the presence of type 2 of

BVD virus, subtype 1a of genotype 1 of BVD virus is predominant in UK herds. On the basis of

phylogenetic analysis of viral genome at 5’ untranslated region, subtype 2a of BVD virus was

recognized and this was similar to that of low virulent US strain of type 2 of BVD virus which

was also verified by monoclonal antibodies (Wakeley et al., 2004). Reverse transcription-

polymerase chain reaction (RT-PCR) was used to identify BVD virus from diarrheal stools,

intestine and bovine abortuses. The positive samples were also tested by virus isolation. The

positive samples were sequenced on 5’UTR and analyzed. A total of 4 viruses (two bovine

abortuses, one intestine, and one diarrheal stool) were isolated by RT-PCR. One BVD virus

isolated from bovine abortuses was biotyped as cytopathic and all other 3 were accepted as non-

cytopathic. Out of 4 isolates, 3 were of genotype 1 and one diarrheal stool isolate was identified

as type 2 of BVD virus. Furthermore, the type 2 of BVDV showed more similarity with that of

found in North American strains than Asian strains (Park et al., 2004). Single tube TaqMan

based RT-PCR assay was used to classify BVD virus into genotypes. Bovine viral diarrhea virus

was quantified by ABI PRISM 7700 sequence detection system and 2 flourogenic probes for 5’

UTR. Serial 10 fold dilutions of RNA were made and sensitivity of the assay was established and


33

compared with standard RT-PCR and 2 tubes TaqMan assay. Single tube assay was found 10 to

100 times more sensitive than 2 tube TaqMan assay and standard RT-PCR. The single tube assay

was also found rapid, sensitive and specific for detection, quantification and classification of

BVD virus (Bhudevi and Weinstock, 2001). To evaluate the proficiency of current methods used

in various diagnostic labs, for the detection of BVD virus, a total of 4 samples (2 negative, one

PI and other with undetectable amount of virus in serum by virus isolation) were submitted to 23

labs. Samples submitted were serum for AC-ELISA, RT-PCR and VI, whole blood for RT-PCR,

VI, skin of ELISA and IHC. Among all the assays, AC-ELISA on skin biopsies revealed

maximum uniformity in detecting positive among labs. RT-PCR and IHC correctly identified

around 85% BVDV positive samples while VI using serum showed poor consistency and lowest

level of agreement. The finding of this study suggested a need for standardization of test methods

(Edmondson et al., 2007).

CHAPTER 3

34

MATERIALS AND METHODS

3.1. COLLECTION OF SAMPLES.

During a period from February 2009 to October 2009, a total of 469 samples (both serum and ear

notch biopsy from each animal) were collected from 12 dairy cattle farms (farms names were

coded to maintain owner confidentiality) located at Prince Edward Island Canada to determine

the prevalence of BVDV persistency. The ear notcher (medium, u-cut, Livestockcocepts, Inc.)

was procured for collection of ear notch biopsies. The instrument was disinfected after collection

of each sample with 10% liquid bleach to prevent the chances of carry over virus contamination.

In this project, the suitability of ear notch biopsy and serum samples for the confirmation

of persistent infection was compared through Real time RT-PCR. Various diagnostic approaches

were also compared using ear notch biopsies. Complete history (age, number and breed of the

animals on a farm, pregnancy status, and previous disease if any), was also noted. The positive

animals were re-sampled after 30 days of initial screening, to differentiate between transient and

persistent infections. The samples were transported to the research Virology Laboratory, Atlantic

Veterinary College, University of Prince Edward Island, Canada for further processing. In the

laboratory, the samples were divided into two groups (A and B) depending upon the age of the

animals. The samples of animals under or equal to 6 months of age were designated as “A” and

other of over 6 months of age as “B”. The groups were made due to initial screening test (serum

neutralization test), the reason being, that normally, P.I. animals older than 6 months of age, are

accompanied by absence of specific BVDV antibodies due to immune tolerance (McClurkin et

al., 1984). Animals below 6 months of age can have passive antibodies in the course of

persistency, if the mother passed the virus to fetus in the course of transient infection and was not

herself persistently infected, so they could not be pre-screened by serology.


35

Each of the ear notch biopsies was incised into five parts (EN1a, EN1b, EN1c, EN1d and EN1e)

with disposable sterile blade. All the sera and EN1a parts of ear notch biopsies of group A (under

6 months of age), were subjected to Real time RT- PCR to compare the diagnostic suitability of

both type of samples, while the samples of animals in group B (older than 6 months of age), were

initially screened by serum neutralization test (SNT). Only those samples of group B, which had

SN titre less than or equal to 1:64 were subjected to Real time RT-PCR (Table 1).

Further, to compare the efficacy of various diagnostic approaches for the detection of

BVDV persistent infection, antigen capture ELISA, and immunohistochemistry assay, were

applied on each respective part of ear notch biopsies of both A and B groups, and compared with

the standard of virus isolation test (Table 1).

3.2. SCREENING OF SAMPLES BY SERUM NEUTRALIZATION TEST (SNT)

The neutralizing antibodies against BVDV were determined through microtitre SN test according

to the OIE prescribed protocol (OIE Terrestrial Manual, 2008) using NADL, a cytopathogenic

BVD viral strain.

3.2.1. PREPARATION OF MADIN DARBY BOVINE KIDNEY (MDBK) CELL

MONOLAYER

The BVDV free MDBK cell line (acquired from University of Guelph, Canada) was maintained

in the research laboratory, Pathology and Microbiology Department, Atlantic Veterinary

College, University Of Prince Edward Island, Canada. The cells were transferred to 25 cm2 carrel

flasks containing 5 ml of Minimum Essential Medium (MEM) (Sigma-Aldrich Co, M-0643) and

incubated at 37°C in CO2 incubator. The flasks having confluent monolayer of adherent cells

were processed for harvesting and transferring to new culture vessels. The growth medium


36

overlying the cell monolayer was poured off aseptically and the monolayer was rinsed, washed

with 3 ml phosphate buffered saline (PBS: pH 8.0) and covered with 2 ml sterile 0.25% trypsin-

EDTA solution (Appendix 1). The mixture was allowed to react on the monolayer for few

minutes at room temperature. The monolayer was periodically observed under an inverted

microscope for rounding and detachment of cells. The trypsin-EDTA solution was removed

quickly to avoid wastage of cells. Four ml of complete MEM (10% horse serum free of BVDV

contamination) was added and mixed to form homogeneous cell suspension. Equal volume of

cell suspension added to each of the 2 carrel flasks already containing 4 ml of growth medium

with 10% horse serum (Sigma-Aldrich Co, H-1270). The whole process was carried out under

aseptic conditions. The flasks were incubated at 37°C and the cells started multiplying. The

complete monolayer was formed in 48 hours (Fig. 4).

Prior to further proceeding, the cells were tested by Real-time RT-PCR to rule out BVDV

contamination that could have occurred through contaminated serum or cells. After confirmation

of BVDV free status, these cells were used for determination of TCID50 of BVDV (NADL),

Virus isolation and titration of neutralizing antibodies.

Dividing Cells Confluent Cells

Fig. 4: MDBK CELLS DURING DIVIDING AND CONFLUENCY SATGE


37

3.2.2. PREPARATION OF MDBK CELL MONOLAYER IN 96- WELL PLATE

Cells suspension, sufficient to form monolayer of cells in 96 well plates was optimized before

seeding the wells. Briefly, monolayer of cells from T-25cm2 carrel flasks was suspended in 10

ml of complete MEM after trypsinisation. The cell suspension was further tenfold diluted by

adding 1ml of cell suspension into 9 ml of MEM before inoculating the plate. A volume of 100

μl of this cell suspension was used in each well to get monolayer of cells within 72 hrs.

3.2.3. TITRATION OF NADL

A microtiter viral titration assay in 96-well plate was used to determine the viral infectivity.

Serial tenfold dilutions of NADL (CP) strain of BVDV was made in Hanks Balanced Salt

Solution (HBSS, Cat # 14025092, GIBCO) up to 11th tube. Fifty microliter (50 μl) of each

dilution of virus was pipetted into each of the eight wells of 96 well plate containing 60%

confluent monolayer of cells. The wells of 12th

column were served as negative virus control

(received HBSS only). The plates were then incubated at 37°C for 5 days. The infectivity for the

CP strain was detected by daily observation of cytopathology with an inverted light microscope,

with the final reading done on day 5. A visible cytopathology in a well was considered indicative

for infectivity. The 50% tissue culture infective dose (TCID50) was calculated according to Reed

and Muench method (Reed and Muench, 1938). Stock virus suspension was diluted to contain

one hundred TCID50 /50 μl and was used for SNT.

3.2.4. SERUM NEUTRALIZATION ASSAY

1. The serum samples were heat-inactivated for 30 minutes at 56°C before use.


38

2. Serial two fold dilutions of the test sera were prepared using MEM as diluent in a cell-culture

grade flat bottomed 96 well microtitre plates. Two wells were used for each dilution of a sample.

3. An equal volume (50 μl) of dilution of BVDV stock, containing 100 TCID50 was added to

each well. Three controls (cell control-HBSS only, virus control- NADL without serum, and 100

TCID50 back titration in four wells) were also included along with each run to validate the assay.

4. The plates were incubated for 60 minutes at 37°C.

5. MDBK monolayer of cells from 25cm2

flask was trypsinised and cell suspension was prepared

according to previously optimized volume as described in 3.2.2. Briefly, 100 μl of cell

suspension was added to each well of the microtitre plates and incubated at 37°C for 5 days in a

5% CO2 atmosphere.

6. The wells were observed microscopically for CPE.

The highest dilution of the serum, inhibiting the cytopathogenic effects of the virus in at least

one of the two replicates was taken as SN titre of each serum sample.

3.3. REAL TIME RT- PCR

3.3.1. SPECIFICITY OF PROBES

To determine probes specificity before proceeding to field samples, 2 BVD virus control strains

(BVDV1-NADL, BVDV2-125c) were tested in the real-time PCR assay. Previously described,

primers and probes were used (Baxi et al., 2006). In the first reaction, TaqMan FAM probe 1 was

tested against NADL, 125c and water (non-template control). In the second reaction, the

templates used in reaction first were tested with TaqMan Quasar probe 2.


39

3.3.2. SENSITIVITY OF THE ASSAY

The detection limit of Real time RT-PCR was determined by making serial 10 fold dilutions of

reference virus strains (BVDV1-NADL and BVDV2-125c) in MEM, based on the infectious titre

of the virus. Various dilutions of stock viruses ranging from 10-1

TCID50 to 10-7

TCID50 were

made and RNA extracted from each dilution was tested through Real time RT-PCR.

3.3.3. TESTING OF FIELD SAMPLES

EN1a part of all ear notch biopsies (EN1a) and sera of group A and selected ones of group B

were subjected to Real time RT PCR to compare the diagnostic suitability of both samples.

3.3.3.1. EXTRACTION OF TOTAL RNA FROM EAR NOTCH BIOPSIES

Total RNAs from ear notch biopsies were extracted using Qiagen RNeasy Mini Kit (QIAGEN,

Cat # 74106) according to manufacturer recommendations. Briefly:

1. A piece (30 mg) of each ear notch biopsy was weighed and homogenized by adding 600 µl of

buffer RLT (β-ME added) in a microcentrifuge tube

2. The tissue lysate was transferred to QIA shredder spin column placed in a 2 ml collection

tube, and centrifuged at 12000 rpm for 2 min.

3. 600 µl of 70% ethanol was added to the cleared lysate, and mixed well by pipetting.

4. 700 μl of the sample was transferred to an RNeasy mini spin column placed in a 2 ml

collection tube and centrifuged at 12000 rpm for 15 sec. Flow through was discarded.

5. 350 μl of buffer RW1 was added onto the RNeasy column and centrifuged for 15 sec at

maximum speed and flow-through was discarded.


40

6. 80 µl of DNase and RDD mixture (10µl DNase and 70µl RDD buffer) was poured onto spin

column membran

Arfan Ahmad - prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1326/1/2438S.pdf · ARFAN AHMAD, have been found satisfactory and recommend that it be processed for the evaluation

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