Robert Barigye

A STUDY OF VIRAL TISSUE TROPISM AND CYTOKINE

EXPRESSION IN BOVINE EPHEMERAL FEVER

ROBERT BARIGYE

BVetMed, MSc, PhD, MANZCVS (Path)

A thesis submitted for the degree of Master of Philosophy at

The University of Queensland in February 2020

School of Veterinary Science ABSTRACT

While the clinical and pathological aspects of Bovine Ephemeral Fever (BEF) have been studied in cattle, major gaps still remain in our understanding of the pathogenesis of the disease. In particular, the cytokine networks that underlie fever and inflammation during acute BEF, and mechanisms that mediate the nascent adaptive immune response have not been empirically defined. In addition, the potential in vivo replication sites of BEFV, and the mechanistic events that underlie the paresis and chronic paralysis in some field BEF cases have also not been defined. The objectives of this research were: (1) to characterise the plasma kinetics of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and IL-10 during natural BEFV infections in cattle; (2) to evaluate the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition; and (3) to determine the overall tissue tropism and potential replication sites, along with assessing the likelihood of neurotropism of BEFV in naturally infected cattle. For the cytokine expression studies, experiments were carried out on two different cohorts of animals raised at two farms at different time points. Briefly, plasma from three BEFV-infected and three uninfected cattle was tested by cytokine-specific cELISA, viraemia monitored by qRT-PCR, and virus neutralising antibody titres determined using a standard protocol. Similarly, plasma from another four virus-infected and uninfected negative control animals was tested for the four study cytokines (IL-2, IFN-γ, IL-6, and IL-10), and viraemia and virus neutralising antibody titres determined. Biological specimens from nine adult cattle that died or were euthanised at different time points following natural BEFV infections were tested for viral antigen and RNA by IHC and qRT-PCR, respectively. In addition, virus isolation in autogenously derived splenic and haemal node cultures, and electron microscopic examination of ultrathin

i sections from various tissues taken from the steer necropsied seven days after BEF diagnosis were performed. For the neurotropism studies, fresh brain, spinal cord, peripheral nerve, and other tissues were collected from four paralysed and six asymptomatic but virus-infected cattle and tested for viral RNA by qRT-PCR.

Formalin-fixed tissues were routinely evaluated for histomorphological lesions and for viral antigen presence and distribution by IHC. Unlike the negative controls, plasma concentrations of IL-1β, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of these heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1β was the most strongly expressed cytokine, IL-6 and

IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, it resolved up to three days after seroconversion. In the second cohort of study cattle, unlike the negative controls, plasma IL-6 and IL-10 were increased in all the virus-infected animals starting several days prior to initiation of viraemia. In one animal, plasma IL-2 and IFN-γ levels were consistently higher than in the other three virus-infected animals.

Notably, this animal had the shortest viraemia while the heifer with the lowest IL-

2/IFN-γ levels demonstrated the longest viraemia. For the virus-tissue tropism studies, BEFV antigens were detected in several tissues/organs in all the study cattle and both virus antigen and RNA were simultaneously demonstrated in the spleen and/or haemal node from 7/9 cattle; the longest period of RNA detection was in the haemal node 120 days following the initial BEF diagnosis. Overall, viral proteins

ii were distributed intracytoplasmically within cells morphologically consistent with macrophages, neutrophils, dendritic cell-like cells and spindle shaped cells with a perivascular location. While definitive identification of the antigen-laden cells would have been informative, attempts at immunophenotyping were fruitless presumably due to antigen denaturation as the tests were done long after completion of the major experiments. In the case study steer necropsied within seven days from the day of definitive BEF diagnosis, viral RNA was detected in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in 13 spleen-derived cell cultures. In this animal, BEFV was isolated from autogenously derived splenic primary cell cultures six days after cessation of viraemia, and characteristic bullet-shaped virions confirmed in the haemal node by TEM. The neurotropism of BEFV was confirmed in the brain and peripheral nerves by IHC, while peripheral neuropathy was histologically demonstrated in three paralysed but not the six virus-infected animals not showing neurological signs. Wallerian degeneration (WD) was present in the lumbar spinal cord of a paralysed steer and in the cervical and thoracic spinal cord segments of three paralysed animals. While no spinal cord lesions were seen in the steer euthanised within seven days of illness, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. The present data documented variable increase in plasma IL-1β, IL-6,

TNF-α, and IL-10 during natural BEFV infections with apparent upregulation of all but

TNF-α appearing to precede seroconversion. Considering the anti-inflammatory properties of IL-10, its upregulation suggests a probable modulatory role that may potentially antagonise the fever response in animals acutely infected with BEFV. As

iii the increase in plasma IL-6 and IL-10 appeared to precede seroconversion, the two cytokines may influence immunological events that pave way to B-cell activation and seroconversion. While remarkable variability in IL-2 and IFN-γ expression was seen in virus-infected animals, the increase in plasma levels of the two cytokines was associated with a shorter viraemia suggesting that, in addition to virus neutralising antibodies, IL-2 and IFN-γ-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Based on these data, BEFV appears to have preferential tropism for bovine lymphoid tissues, and the spleen and haemal node may particularly be potential in vivo sites for BEFV replication. The present data also confirm that BEFV is neurotropic in cattle and that, in addition to WD in the spinal cord, peripheral neuropathy and non-suppurative brain stem encephalitis may contribute to chronic paralysis seen in BEFV-infected downer cattle. Additional studies are needed to define the precise role of T cells in anti-BEFV adaptive immune responses. The potential in vivo replication sites of the BEFV in cattle could be further characterised by in situ hybridisation and other molecular tools.

DECLARATION BY AUTHOR

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others towards the jointly- authored works that I have included in this thesis.

I have also clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis does not include any work that has previously been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution.

I acknowledge that an electronic copy of my thesis must be lodged with the

University Library and, subject to the policy and procedures of The University of

Queensland, the thesis may be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the

Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate, I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

PUBLICATIONS THAT ARE INCLUDED IN THE THESIS

Per UQ Authorship Policy (PPL 4.20.04 Authorship), contributions of all co-authors to the above publications are briefly described in the table below.

I) Publication citations – incorporated as Chapter 2.

BARIGYE, R., MELVILLE, L.F., DAVIS, S., WALSH. S., HUNT, N., HUNT, R.,

ELLIOTT, N. 2015. Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle. Veterinary Immunology and Immunopathology 168:159-163.

BARIGYE, R., MELVILLE, L.F., DAVIS, S., WALSH, S., HUNT, N., HUNT, R. 2016.

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus. Veterinary

Microbiology 186:111-116.

Contributor Statement of contribution

Robert Barigye (Candidate) Designed experiments (80%)

Manuscript writing and editing (90%)

Lorna Melville Designed experiments (20%)

Manuscript writing and editing (3%)

Steve Davis Designed experiments (0%)

Manuscript writing and editing (2%)

Susan Walsh Designed experiments (0%)

Manuscript writing and editing (2%)

Neville Hunt Designed experiments (0%)

Manuscript writing and editing (1%)

Roslyn Hunt Designed experiments (0%)

Manuscript writing and editing (1%)

Nikki Elliott Designed experiments (0%)

Manuscript writing and editing (1%)

III) Publication citations – incorporated as Chapter 3.

BARIGYE, R., DAVIS, S., HUNT, R., HUNT, N., WALSH, S., ELLIOTT, N.,

DYRTING, K., WEIR, R., MELVILLE, L.F. 2017. Post-viraemic detection of bovine ephemeral fever virus by use of autogenous lymphoid tissue-derived bovine primary cell cultures. Australian Veterinary Journal. 95:49-52.

BARIGYE, R., BURNUP, C., DAVIS, S., AUMANN, S., HUNT, R., HUNT, N.,

WALSH, S., MELVILLE, L.F. 2016. Application of immunohistochemistry and quantitative real time polymerase chain reaction to the study of tissue virus tropism in adult cattle infected with the bovine ephemeral fever virus. Journal of Clinical

Experimental Pathololgy. 6:3, (Suppl)

MELVILLE, L.F., BARIGYE, R., DAVIS, S., HUNT, R., HUNT, N., WALSH, S.,

ELLIOTT, N., BURNUP, C., AUMANN, S., DAY, C., DYRTING, K., WEIR, R. 2016. A study of viral tissue tropism and potential replication sites of the bovine ephemeral fever virus in cattle. 12th Symposium of the Mosquito Control Association of Australia and Arbovirus Research in Australia Surfers Paradise, Queensland 2016.

MELVILLE, L.F., BARIGYE, R., BURNUP, C., DAVIS, S., AUMANN, S., HUNT, R.,

HUNT N., WALSH S. 2016. Evaluation of potential in vivo replication sites for bovine ephemeral fever virus in naturally infected cattle. 12th Symposium of the Mosquito

vii

Control Association of Australia and Arbovirus Research in Australia Surfers

Paradise, Queensland 2016.

Contributor Statement of contribution Robert Barigye (Candidate) Designed and conducted experiments (80%) Manuscript writing and editing (85%) Lorna Melville Designed experiments (15%) Manuscript writing and editing (3%) Steve Davis Designed and conducted experiments (5%) Manuscript writing and editing (2%) Susan Walsh Designed and conducted experiments (0%) Manuscript writing and editing (2%) Neville Hunt Designed experiments (0%) Manuscript writing and editing (1%) Roslyn Hunt Designed experiments (0%) Manuscript writing and editing (1%) Nikki Elliott Designed and conducted experiments (0%) Manuscript writing and editing (1%) Catherine Burnup Designed and conducted experiments (0%) Manuscript writing and editing (1%) Susan Aumann Designed and conducted experiments (0%) Manuscript writing and editing (1%) Cheryl Day Designed experiments (0%) Manuscript writing and editing (1%) Kitman Dyrting Designed experiments (0%) Manuscript writing and editing (1%)

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Richard Weir Designed experiments (0%) Manuscript writing and editing (1%)

III) Publication citations – incorporated as Chapter 4.

BARIGYE, R., DAVIS, S., HUNT, R., HUNT, N., WALSH, S., ELLIOTT, N.,

BURNUP, C., AUMANN, S., DAY, C., DYRTING, K., WEIR, R., MELVILLE, L.F.

2016. Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle. Australian

Veterinary Journal. 94:362-370.

Contributor Statement of contribution Robert Barigye (Candidate) Designed and conducted experiments (80%) Manuscript writing and editing (85%) Lorna Melville Designed experiments (15%) Manuscript writing and editing (3%) Steve Davis Designed and conducted experiments (3%) Manuscript writing and editing (2%) Susan Walsh Designed and conducted experiments (1%) Manuscript writing and editing (2%) Neville Hunt Designed experiments (0%) Manuscript writing and editing (1%) Roslyn Hunt Designed experiments (0%) Manuscript writing and editing (1%) Nikki Elliott Designed and conducted xperiments (1%) Manuscript writing and editing (1%) Catherine Burnup Designed experiments (0%) Manuscript writing and editing (1%) Susan Aumann Designed experiments (0%)

Manuscript writing and editing (1%) Cheryl Day Designed experiments (0%) Manuscript writing and editing (1%) Kitman Dyrting Designed experiments (0%) Manuscript writing and editing (1%) Richard Weir Designed experiments (0%) Manuscript writing and editing (1%)

SUBMITTED MANUSCRIPTS INCLUDED IN THESIS

No manuscripts submitted for publication

OTHER PUBLICATIONS DURING CANDIDATURE

CONFERENCE ABSTRACTS:

MELVILLE, L.F., BARIGYE, R., DAVIS, S., HUNT, R., HUNT, N., WALSH, S.,

MELVILLE, L.F., BARIGYE, R., BURNUP, C., DAVIS, S., AUMANN, S., HUNT, R.,

HUNT N., WALSH S. 2016. Evaluation of potential in vivo replication sites for bovine ephemeral fever virus in naturally infected cattle. 12th Symposium of the Mosquito

Control Association of Australia and Arbovirus Research in Australia Surfers

Paradise, Queensland 2016.

BARIGYE, R., BURNUP, C., DAVIS, S., AUMANN, S., HUNT, R., HUNT, N.,

WALSH, S., MELVILLE, L. 2016. Application of immunohistochemistry and quantitative real time polymerase chain reaction to the study of tissue virus tropism

x in adult cattle infected with the bovine ephemeral fever virus. 7th World Congress on

Molecular Pathology July 25-26, 2016 Melbourne, Australia.

CONTRIBUTION BY OTHERS TO THE THESIS

This thesis is my original work and is based on part of the studies that were conducted under a large research project at the BVL in Darwin, NT. A number of people including Dr Lorna Melville - the principal investigator (PI) of the BEF research project - and the technical personnel at the BVL made some contributions towards the implementation of the research project part of which was the basis for my thesis. Dr. Lorna Melville was the PI of the BEF research project and wrote the original grant proposal from which my fellowship was funded. In addition to providing overall guidance and leadership during the implementation phase of the research project, Dr Melville also served as my Darwin-based immediate supervisor. With the exception of Cheryl Day who relocated to the University of Adelaide half way the project, co-authors Steve Davis, Susan Walsh, Neville Hunt, Roslyn Hunt, Nikki

Elliott, Catherine Burnup, Sue Aumann, and Cheryl Day are technicians at the BVL.

As such, these individuals contributed to the laboratory testing of samples as part of the BEF research project. For example, as virology technician, Steve Davis contributed to the virus neutralising assays, and as molecular technicians, Susan

Walsh and Nikki Elliott contributed to the testing of samples by qRT-PCR. Rose Hunt and Neville Hunt provided support in collecting blood samples from the sentinel cattle. Dr. Kitman Dyrting is a veterinary pathologist at the BVL and participated in 1 out of the 10 research necropsy examinations that were performed as part of this research. On other hand, Dr. Richard Weir is a Senior Virologist at the BVL and

xi helped with coordinating the evaluation of ultrathin lymphoid tissue section by transmission electron microscopy at the Charles Darwin University.

A/Prof Chiara Palmieri contributed to the revision of the histological and immunohistochemical slides.

STATEMENT ON PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE

AWARD OF ANOTHER DEGREE

No works submitted towards another degree have been submitted towards another degree have been included in this thesis.

RESEARCH INVOLVING HUMAN OR ANIMAL SUBJECTS

Conduction of the research presented in this thesis involved cattle but not human subjects. Note that all experimental work on the cattle was approved by the Charles

Darwin University Animal Ethics Committee (CDU-AEC) through Approval letter No.

A12036 – A study of cytokine responses and viral tissue tropism in bovine ephemeral fever; and Approval letter No. A14001 – A study of CD4+ and CD8+ lymphocyte responses in bovine ephemeral fever virus infections in cattle

(Appendices 1 and 2 respectively). Note that the approvals from the CDU-AEC were duly ratified by the University of Queensland Animal Ethics committee (UQ-AEC).

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ACKNOWLEDGEMENTS

I would like to thank Dr. Chiara Palmieri, my UQ-based principal thesis advisor, and the entire advisory committee (Dr. Helen Owen, Dr. Simmon Gregory, and Dr. Toby

St George) for the guidance and mentorship during the time I worked on this thesis. I am particularly indebted to Dr. St George for allowing be unlimited access to his archives and for providing insightful feedback throughout the time I was conducting the research work.

I am greatly indebted to Dr Lorna Melville, my Darwin-based supervisor for the exceptional mentorship and moral support provided during my time at the BVL.

Without her vision, leadership and support, realization of this thesis would not have been possible.

Let me also extend my sincere gratitude to the entire technical team at the BVL

(Steve Davis, Susan Aumann, Catherine Burnup, Nikki Elliott, Susan Walsh, Cheryl

Day, Rose Hunt, and Neville Hunt) for the unfailing support throughout the duration of the BEF research project. My special thanks also go to my pathologist colleagues

Drs Cathy Shilton and Kitman Dyrting, and to senior virologist Dr. Richard Weir for useful insights and comradeship that so much contributed to the success of the thesis research work.

The Meat Livestock Australia is acknowledged for funding this research through grant No. B.STU.0246 while the NTG and the DPIR are also appreciated for the financial support during the memorable time I spent at the BVL. There is no doubt that the financial resources invested towards the implementation of the BEF

xiii research project have helped to advance our understanding of the pathogenesis of the economically important cattle disease that BEF is.

Last but not least, let me thank my wife Esther for taking care of our children during the time I was away doing this work. To my mother, I unreservedly thank you for your constant prayers, and to my late father you are heartily remembered for inculcating in me the love of education at a very young age.

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KEYWORDS

Bovine ephemeral fever, cattle, cytokines, lymphoid tissues, neurotropism, peripheral neuropathy, tissue tropism

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 070709, Veterinary Pathology, 50%

ANZSRC code: 070705, Veterinary Immunology, 40%

ANZSRC code: 070712, Veterinary Virology, 10%

Fields of Research (FoR) Classification

FoR code: 0707, Veterinary Sciences, 100%

TABLE OF CONTENTS

Abstract …………….…………………………………………………………… i

Declaration by author …………….…………………………………………… v

Publications included in the thesis ……………………………………………… vi

Other publications during candidature ………………………………………… x

Contributions of others to the thesis …………………………………………. xi

Statement on parts of the thesis submitted to qualify for the award of another degree ……………………………………………………………….. xii

Research involving human or animal subjects …………………………….. xii

Acknowledgements ……………………………………………………………. xiii

Key words………………………………………………………………………… xv

Australian and New Zealand Standard Research Classification (ANZSRC).. xv

Table of contents………………………………………………………………… xvi

List of Figures……………………………………………………………………… xxi

List of Tables……………………………………………………………………. xxv

Virus Abbreviations……………………………………………………………… xxvi

General Abbreviations…………………………………………………………… xxvi

Chapter 1 – Introduction and Literature Review…………………………… 1

1.1 BACKGROUND…………………………………………………………….. 1

1.2 AETIOLOGY OF BEF……………………………………………………… 2

1.3 HOST RANGE OF BEFV………………………………………………….. 5

1.4 ARTHROPOD VECTORS OF BEFV……………………………………. 7

1.5 PATHOGENESIS OF BEF………………………………………………… 8

1.5.1 Clinical and pathological features………………………………. 8

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1.5.2 Role of cytokines in the pathogenesis of BEF.………………… 9

1.5.3 Tissue tropism of BEFV…………………………………………… 10

1.5.4 Potential neurotropism of the BEFV…………………………….. 11

1.6 MECHANISMS OF IMMUNITY IN BEF…………………………………. 12

1.6.1 Humoral immunity…………………………………………………. 12

1.6.2 Cell-mediated immunity…………………………………………… 14

1.7 CONCLUDING REMARKS……………………………………………… 15

1.8 AIMS OF THE STUDY…………………………………………………… 17

Chapter 2 – Kinetics of the virus neutralising antibody response and selected plasma cytokines during acute disease and the innate- adaptive immune response transition in cattle naturally infected with the bovine ephemeral fever virus…………………………………………… 19

2.1 ABSTRACT…………………………………………………………………. 20

2.2 INTRODUCTION…………………………………………………………… 22

2.3 MATERIALS AND METHODS……………………………………………. 24

2.3.1 Study animals……………………………………………………… 24

2.3.2 qRT-PCR for BEFV……………………………………………… 26

2.3.3 Virus neutralisation test…………………………………………… 27

2.3.4 Immunoenzymatic assays for bovine cytokines……………… 28

2.4 RESULTS ………………………………………………………………….. 29

2.4.1 Study cattle – cohort 1 …………………………………………… 29

2.4.1.1 Fever, seroconversion, and resolution of viraemia …… 29

2.4.1.2 Kinetics of plasma cytokines (IL-1β, IL-6, TNF-α, IL-

10) ……………………………………………………….. 32

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2.4.2 Study cattle—cohort 2…………………………………………….. 33

2.4.2.1 Viraemia and the kinetics of virus neutralising antibody

response …………………………………………………………. 33

2.4.2.2 Kinetics of plasma IL-10, IL-6, IL-2, and IFN-γ ……… 36

2.5 DISCUSSION ………………………………………………………………. 40

2.6 CONCLUSIONS……………………………………………………………. 48

Chapter 3 – Tissue tropism and potential replication sites of the bovine ephemeral fever virus in cattle……………………………………… 51

3.1 ABSTRACT………………………………………………………………… 52

3.2 INTRODUCTION…………………………………………………………… 53

3.3 MATERIALS AND METHODS…………………………………………… 55

3.3.1 Experimental cattle ………………………………………………. 55

3.3.2 Virus neutralisation test and virus isolation ……………………. 56

3.3.3 qRT-PCR for BEFV ……………………………………………….. 57

3.3.4 Tissue processing and histology ………………………………… 57

3.3.5 Autogenously-derived splenic and haemal node cell cultures 58

3.3.6 Paraffin-embedded block of virus infected cell cultures ………. 59

3.3.7 Immunohistochemistry …………………………………………… 60

3.3.8 Transmission electron microscopy ……………………………… 61

3.4 RESULTS …………………………………………………………………... 62

3.4.1 Virus neutralisation test and virus isolation…………………… 62

3.4.2 qRT-PCR results ………………………………………………… 63

3.4.3 Histopathology …………………………………………………….. 67

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3.4.4 Detection of BEFV in autogenous splenic and haemal node

cell cultures……………………………………………………….. 67

3.4.5 IHC detection of BEFV proteins in BSR and MDBK cell lines .. 69

3.4.6 IHC test for BEFV proteins in bovine tissues ………………….. 73

3.4.7 Transmission electron microscopy …………...... 81

3.5 DISCUSSION………………………………………………………………. 81

3.6 CONCLUSIONS 86

Chapter 4 – Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle…………………………………………………………………… 87

4.1 ABSTRACT………………………………………………………………… 88

4.2 INTRODUCTION…………………………………………………………… 89

4.3 MATERIALS AND METHODS…………………………………………… 90

4.3.1 Study animals and clinical monitoring…………………………… 90

4.3.2 qRT-PCR for BEFV……………………………………………… 90

4.3.3 Virus neutralisation test and virus isolation ……………………. 91

4.3.4 Necropsy examination of BEFV-infected cattle………………… 91

4.3.5 Tissue processing and histological evaluation………………… 92

4.3.6 IHC test for BEFV antigen.……………………………………… 93

4.4 RESULTS ………………………………………………………………….. 93

4.4.1 Animals and clinical monitoring………………………………….. 93

4.4.2 qRT-PCR results………………………………………………… 94

4.4.3 Virus neutralisation test and virus isolation results …………… 95

4.4.4 Neuropathological and other histological abnormalities……… 101

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4.4.5 Results of IHC test on nervous tissues………………………….. 108

4.5 DISCUSSION …………………………………………………………….. 113

4.6 CONCLUSIONS…………………………………………………………… 117

Chapter 5 – General Discussion………………………………………………. 119

5.1 The role of cytokines in the immunity and pathogenesis of

BEF……………………………………………………………………….. 119

5.2 Clarification of the tissue tropism and potential in vivo replication sites

of BEFV……………………………………………………………………… 121

5.3 Clarification on the neuropathology and possible neurovirulence of

BEFV………………………………………………………………………… 123

Chapter 6 – Bibliography………………………………………………………. 126

APPENDIX 1 – Animal Ethics Approval No 1…………………………….. 148

APPENDIX 2—Animal Ethics Approval No 2……………………………. 150

APPENDIX 3—Raw qRT-PCR data ……………………………………… 151

LIST OF FIGURES

Figure 1.1: Structure and morphology of BEFV 4

Figure 2.1: Comparative kinetics of plasma cytokines (IL-1β, IL-6, IL-10,

and TNF-α) in three adult cattle naturally infected with BEFV

and three uninfected age-matched negative

controls…………………………………………………………… 32

Figure 2.2: Relationship between mean rectal temperature and mean

plasma concentrations of IL-1β, TNF-α and IL-6 in adult cattle

naturally infected with BEFV………………………………… 33

Figure 2.3: Kinetics of plasma IL-10 in four adult Brahman cattle infected

with BEFV and four uninfected negative controls ………… 36

Figure 2.4: Kinetics of plasma IL-6 in four adult Brahman cattle infected

with BEFV and four uninfected negative controls………… 37

Figure 2.5: Kinetics of plasma IL-2 in four adult Brahman cattle infected

with BEFV and three uninfected negative controls……………. 39

Figure 2.6: Kinetics of plasma IFN-γ in four adult Brahman cattle infected

with BEFV and three uninfected negative controls………... 40

Figure 3.1 Photomicrograph of splenic-derived cell cultures from the 63

steer euthanased 6 days following a BEFV infection diagnosis

Figure 3.2: IHC for the detection of BEFV antigen on sections of paraffin-

embedded virus-infected and uninfected BSR

cells………………………………………… 70

Figure 3.3: IHC for the detection of BEFV antigen on sections of paraffin-

embedded BSR cells infected with KIMV and BRMV…………. 71

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Figure 3.4: IHC for the detection of BEFV antigen on sections of paraffin-

embedded BSR cells infected with ADRV and BTV-1……….. 72

Figure 3.5: IHC for the detection of BEFV antigen in sections of paraffin-

embedded BSR cells infected with AKAV and BoHV-1……… 73

Figure 3.6: IHC for the detection of BEFV antigen on a section of spleen

collected from a Brahman steer euthanised 1 week following

the initial BEF diagnosis………………………... 75

Figure 3.7: IHC for the detection of BEFV antigen on a section of spleen

collected from a Droughtmaster steer euthanised 93 days

following the initial BEF diagnosis……….. 76

Figure 3.8: IHC for the detection of BEFV antigen in a section of haemal

node collected from a Brahman steer euthanised 1 week

following the initial BEF diagnosis………... 77

Figure 3.9: IHC for the detection of BEFV antigen in a section of lung

collected from a Brahman steer euthanised 1 week following

the initial BEF diagnosis………………………... 78

Figure 3.10: IHC for the detection of BEFV antigen on a section of synovial

membrane (synovial joint) collected from a Brahman heifer

euthanased 23 days following the initial BEF diagnosis 79

……….

Figure 3.11: IHC for the detection of BEFV antigen on a section of liver

collected from a Brahman heifer euthanased 23 days

following the initial BEF 80

diagnosis…………………………………………

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Figure 4.1: Peripheral nerve collectedfrom the left brachial plexus of a

paralysed BEFV-infected downer steer (H&E)…………………. 104

Figure 4.2: Peripheral nerve collected from the left brachial plexus of a

paralysed BEFV-infected downer steer showing abundant

Alcian blue-reactive myxomatous nerve tissue debris

surrounding remnants of a unremarkably staining nerve

…………………………………………………………………….. 105

Figure 4.3: Peripheral nerve collected from the left brachial plexus of a

paralysed BEFV-infected downer steer (animal no. 3)

showing extensive areas of myelin-negative myxomatous

degenerate nerve tissue debris surrounding the normalpart of

the nerve (Luxol fast blue)……………………………………. 106

Figure 4.4: Section of the left gluteal nerve of a BEFV-infected heifer

without neurological signs (animal no. 10) showing abundant

myelin staining throughout the nerve tissue (Luxol fast blue)… 107

Figure 4.5: A. Section of the left gluteal nerve from a BEFV-infected

heifer (animal no. 5) stained with the mAb DB5 and

counterstained with Mayer’s haematoxylin showing a few

Schwann cells with intracytoplasmic viral antigen; B. Left

gluteal nerve from a BEFV-infected heifer (animal no. 5)

incubated with negative control mouse serum and

counterstained with Mayer’s haematoxylin, showing

remarkable lack of immunostaining…………………………….. 109

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Figure 4.6 Gluteal nerve from a BEFV-negative animal stained with the

mAb DB5 and counterstained with Mayer’s haematoxylin and

showing absence of BEFV antigen……………………………. 110

Figure 4.7 Peripheral nerve collected from the right brachial plexus of a

chronically paralysed BEFV-infected downer steer (animal no.

3) stained with the mAb DB5 and counterstained with Mayer’s

haematoxylin……………………………………………………….. 111

Figure 4.8 A. Section of medulla oblongata from a chronically paralysed

BEFV-infected steer (animal no. 6) stained with the mAb DB5

and counterstained with Mayer’s haematoxylin; B. Section of

medulla oblongata from a BEFV-infected chronically

paralysed steer (animal no. 6) incubated with the negative

control mouse serum and counterstained with Mayer’s

haematoxylin, showing lack of viral immunostaining. ………… 112

Figure 4.9 Section of the medulla oblongata from a BEFV-negative steer

that died from listeriosis, incubated with the mAb DB5 and

counterstained with Mayer’s haematoxylin – showing absence

of BEFV antigen…………………………………………………… 113

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LIST OF TABLES

Table 2.1: Primer probes used in the qRT-PCR assay for the detection

of BEFV RNA …………………………….. 27

Table 2.2: Rectal temperatures (◦C) of study cattle during a six-day

monitoring period………………………………………………… 30

Table 2.3: Virus neutralising antibody titres and qRT-PCR data for

three cattle naturally infected with BEFV……………………… 31

Table 2.4: Relationship between the virus neutralising antibody

response and the kinetics of viraemia during natural BEFV

infections in four adult Brahman cattle………………………… 35

Table 3.1: Results of qRT-PCR, virus neutralisation test, and IHC tests

on biological specimens collected from nine adult Brahman

cattle with BEFV infection………………………………………. 65

Table 3.2: Fractions of the different passage levels of spleen and

haemal node-derived cell cultures that were positive for

BEFV RNA by qRT-PCR assay………………………………… 68

Table 4.1: A summary of the data obtained from the neuropathology

study of BEFV-infected cattle ………………………………….. 97

Table 4.2: A summary of the histopathology data obtained from the

neuropathology study of BEFV-infected cattle ………………. 99

xxv

LIST OF ABBREVIATIONS a) Virus name abbreviations

ADRV Adelaide river virus AKAV Akabane virus BEFV Bovine ephemeral fever virus BoHV-1 Bovine herpesvirus-1 BRMV Berrimah virus BTV-1 Bluetongue virus serotype-1 KIMV Kimberly virus KOTV Kotonkan virus MALV Malakal virus OBOV Obodhiang virus PUCV Puchong virus

b) General abbreviations

AEC 3-amino-9-ethylcarbazole substrate BEF Bovine ephemeral fever BSR Baby kidney hamster cell line BVL Berrimah Veterinary Laboratories CD4+ Cluster of differentiation 4+ CD8+ Cluster of differentiation 8+ cELISA Competitive enzyme-linked immunosorbent assay Cox-1 Cyclooxygenase-1 Cox-2 Cyclooxygenase-2 Ct value Cycle threshold value DPIR Department of Primary Industry and Resources ELISA Enzyme-linked immunosorbent assay G1 Antigenic site 1 of the viral envelope glycoprotein of the bovine ephemeral virus

xxvi

G2 Antigenic site 2 of the viral envelope glycoprotein of the bovine ephemeral virus G3 Antigenic site 3 of the viral envelope glycoprotein of the bovine ephemeral virus IFN-α Interferon-α IFN-β Interferon-β IFN-γ Interferon-γ IHC Immunohistochemistry IL-10 Interleukin-10 IL-1β Interleukin-1β IL-2 Interleukin-2 IL-6 Interleukin-6 mAb Monoclonal antibody MDBK Bovine kidney cell line MEM Medium essential medium min Minute NK Natural killer cells NT Northern Territory NY New York

OD450 Optic density reading at 450nm PBS Phosphate buffer saline PCR Polymerase chain reaction

PDE2 Prostaglandin-E2 recT Rectal temperature RNA Ribonucleic acid RT Room temperature RT-PCR Reverse transcriptase polymerase chain reaction TBS/T Tris buffered saline-Tween 20, pH 7.4

TCID50 Tissue culture infectious dose T-cell T - lymphocyte Th1 Type-1 helper cells Th2 Type-2 helper cells

xxvii

TNF-α Tumour necrosis factor-α µg microgram µl microlitre

xxviii

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 BACKGROUND

BEF is an infectious disease of cattle and water buffaloes (Bubalis bubalis) caused by an arthropod-borne rhabdovirus (St George, 1986). The disease in cattle is prevalent in tropical and subtropical regions of Asia, Australia, and Africa (Davies et al., 1990; St George et al., 1995; Walker and Klement, 2015), but has never been reported in North America, South America, Europe, nor in the Pacific islands (St

George et al., 1995; Walker and Klement, 2015). Typically, clinical BEF is characterised by short-lived transient symptoms that include but are not limited to polyphasic fever, somnolence, synovitis, lameness, muscle stiffness and disinclination to move, as well as inappetence (Basson et al., 1970; Mackerras,

1940; St George, 1986). While these symptoms are often dramatic in onset and severity, affected cattle almost invariably demonstrate complete recovery (St

George, 1986). Based on the transient nature of the fever and other clinical signs, the disease in cattle is also referred to as “three-day sickness” in Australia and

Africa, and “bovine epizootic fever” in Japan (St George et al., 1986). In a minority of

BEFV-infected cattle, the disease is also characterised by paresis and/or paralysis

(Basson et al., 1970; Nadi and Negi, 1999; St George et al., 1995; Young and

Spradbrow, 1990a) often requiring euthanasia. These authors also indicate that a very small percentage of affected cattle may suddenly die. In addition, a severe reduction in milk production is seen in milking animals (Davis et al., 1984) and animals that have recovered from BEF take longer to gain weight (St George, 1986) which has negative implications for beef production.

After the onset of the downer syndrome in the minority of BEF infected animals that become paretic, euthanasia becomes inevitable, since other forms of treatment are not helpful. While the mortality rate of BEF is usually less than 1%, morbidity rates of up to 100% and case-fatality rates greater 20% have been reported (Hsieh et al.,

2005; St George, 1986; Tonbak et al., 2013; Zheng et al., 2012). The economic impact of BEF may be linked to severe reduction in milk production in lactating dairy cattle, post-infection intractable loss of condition in beef cattle, and immobilisation of affected buffaloes (Davis et al., 1984; Nandi and Negi, 1999; St George, 1986). BEF may also adversely affect livestock trade since disease free countries or regions will refuse to accept cattle from BEF-endemic areas (Aziz-Boaron et al., 2012). In BEF- endemic areas, the disease control options are limited to vaccination of susceptible cattle with suboptimum inactivated vaccines (St Aziz-Boaron et al., 2015; Walker and

Klement, 2015).

1.2 AETIOLOGY OF BEF

Bovine ephemeral fever virus (BEFV), the aetiological agent of BEF, belongs to the

Genus Ephemerovirus within the Family Rhabdoviridae (Murphy et al., 1972; Walker and Klement, 2015). In addition to BEFV, the genus Ephemerovirus also includes closely related vector-borne rhabdoviruses with all members typically characterised by bullet-shaped virions (Murphy et al., 1972; Blasdell et al., 2012a; Blasdell et al.,

2012b; Walker and Klement, 2015). Of these viruses, those that have been isolated from Australia include the Kimberley virus (KIMV) (Cybinski and Zakrzewski, 1983),

Berrimah virus (BRMV) (Gard et al., 1983), and Adelaide river virus (ARV) (Gard et al. 1984). However, as these viruses have only been isolated from subclinically infected animals, they are not deemed to be pathogenic in cattle. However, KIMV

2 demonstrates close antigenic overlap with BEFV to the extent that it is detectable by

BEFV-specific serum virus neutralisation tests (Cybinski et al., 1992).

Other closely related arthropod-borne rhabdoviruses of interest include Malakal virus

(MALV) (Blasdell et al., 2012b; Calisher et al., 1989), Kotonkan virus (KOTV) (Bauer and Murphy, 1975; Kemp et al., 1973; Blasdell et al., 2012a), Obodhiang virus

(OBOV) (Schimidt et al., 1963; Shope, 1982; Blasdell et al., 2012a), and Puchong virus (PUCV) (Calisher et al., 1989). MALV was first isolated from a mosquito in southern Sudan and strongly cross-reacts with BEFV, KIMV, OBOV, and PUCV

(Calisher et al., 1989). Recent phylogenetic studies have also demonstrated a close genetic relationship between MALV and BEFV (Blasdell et al., 2012b). KOTV was first isolated from pooled midges of the genus Culicoides spp in Nigeria and thereafter was shown to cause a BEF-like clinical illness in cattle (Kemp et al., 1973;

Tomori et al., 1974). However, this virus has not been reported in Australia. On the other hand, OBOV was first isolated in southern Sudan from mosquitoes of the genus Mansonia uniformis (Schmidt et al., 1965) and was later shown to cross-react with KOTV (Bauer and Murphy, 1975; Calisher et al., 1989). While OBOV has not been isolated from Australia, molecular studies have demonstrated a close genetic relationship between this virus and the ADRV (Blasdell et al., 2012a; Bourhy et al.,

2005; Calisher et al., 1989).

Structurally, the BEFV virion is composed of a helical genome surrounded by a matrix protein (M) and a lipid envelope (Murphy et al., 1972; Walker et al., 1991), along with a nucleocapsid made up of four major constituents including a simple genome composed of negative sense ssRNA, a nucleoprotein (N), phosphoprotein

(P), and a multi-functional enzyme (Figure 1.1) (Murphy et al., 1972; Walker et al.,

1991). Anchored within the lipid envelope and projecting towards the exterior of the bullet-shaped virion are several transmembrane glycoproteins designated G1, G2,

G3 and G4 (Walker et al., 1991). Of these, G1, G2, and G3 bear epitopes that are important in stimulating a neutralising antibody response in the vertebrate host

(Cybinski et al., 1990; Uren et al., 1994). While the N protein is also immunogenic in cattle, it does not induce a neutralising antibody response (Uren et al., 1993; Walker et al., 1994).

Figure 1.1: “Structure and morphology of BEFV. (A) Structural organisation of the

14.9 kb BEFV genome shown as being arranged in negative sense. Structural protein genes (N, P, M, G and L) are shown in black and the various accessory genes are coloured. (B) Transmission electron micrograph showing BEFV virions and defective-interfering (DI) particles. Scale bar 100 nm. (C) Structural model of a monomeric subunit of the BEFV G protein derived by homology modelling using the

4 pre-fusion form of the VSV G protein as a template. The model illustrates the three major neutralization sites (G1, G2 and G3a/b) and amino acid residues shown to be under positive selection in Australia (Trinidad et al., 2014)”. Source: Walker and

Klement (2015).

1.3 HOST RANGE OF BEFV

Clinical BEF has been reported in both Bos indicus and Bos taurus breeds of cattle

(Young, 1979) as well as in water buffaloes (Bubalis bubalis) (Bai et al., 1989; Patel et al., 1993). In a study conducted by Bai et al. (1989), the experimental inoculation of BEFV in a water buffalo was associated with a mild disease (Bai et al., 1989), although natural BEFV infections characterised by severe clinical disease have also been reported in water buffaloes (Patel et al., 1993). On the other hand, experimental inoculation of BEFV in sheep was only associated with subclinical infections with few of the animals showing short-lived pyrexia (Hall et al., 1975).

While some of the experimental sheep showed mild haematological changes and developed neutralising antibodies following experimental BEFV infections, efforts to isolate the virus from their peripheral blood were unsuccessful (Hall et al., 1975).

Interestingly, however, specimens of buffy coat prepared from the peripheral blood of experimentally infected sheep induced mild clinical BEF in cattle (Chiu and Lu,

1998). In another study performed in Taiwan, BEFV neutralising antibodies were demonstrated in sheep and goats (Chiu and Lu, 1989). However, research done in

BEF-enzootic regions elsewhere have not shown any evidence of natural BEFV infection in sheep (Aziz-Boaran et all., 2015; Cybinski and Zakrzewiski, 1983;

Doherty et al., 1972). In addition, BEFV neutralising antibodies have been reported in camels (Camelus dromedaries) in northern Africa (Elbaoumy et al., 2013). While

5 most of the above animal species are not thought to present with the clinical disease, an ephemeral fever-like illness has been observed in camels in Somalia and north- eastern Kenya (Dirie and Abdurahmanm, 2003).

Besides domestic animals, evidence of seroconversion to BEFV has been reported in several wildlife species in Africa (Davies et al., 1975; Anderson and Rowe, 1998;

Hamblin et al., 1990; Barnard, 1997), Middle East, Asia (Aziz-Boaron et al., 2012;

Lim et al., 2007) and Australia (Cybinski et al., 1983; McKenzie et al., 1985; St

George, 1986) leading to the conclusion that wildlife species may serve as reservoirs for the disease in cattle (Barnard, 1997; Davies et al., 1975). For example, BEFV neutralising antibodies have been demonstrated in several wild ungulates in Africa, such as the African buffalo (Syncerus caffer), wildebeest (Connochaetes taurinus,

Connochaetes gnou), hartebeest (Alcelaphus buselaphus), topi (Damaliscus korrigum), waterbuck (Kobus ellipsiprymnus), blesbok (Damaliscus dorcas phillipsi), tsessebe (Damaliscus lunatus), springbok (Antidorcus marsupialis), sable antelope

(Hippotragus niger), impala (Aepycerus melampus), kudu (Tragelaphus strepsiceros), eland (Taurotargus oryx), bushbuck (Tragelaphus scriptus) and giraffe

(Giraffa camelopardalis) (Anderson et al., 1998; Barnard, 1997; Davies et al., 1975;

Hamblin et al., 1990). Additionally, evidence of BEFV exposure has been reported in the African elephant (Loxodonta africana), oryx (Oryx beisa), warthog

(Phacochoerus aethiopicus), lechwe (Kobus leche), hippopotamus (Hippopotamus amphibius) and the gazelle (Gazella granti) (Hamblin et al., 1990). Outside Africa, neutralising antibodies against BEFV have also been described in the Persian fallow deer (Dama d. mesopotamica) and gazelle (Gazella g. gazella) in Israel (Aziz-

Boaran et al., 2015)], pigs (Sus scrofa) in South Korea (Lim et al., 2007), and in red

6 deer (Cervus elaphus), Rusa deer (C. timorensis), and Chital deer (Axis axis) in

Australia (Cybinski and Zakrzewiski, 1983; St George, 1986; McKenzie et al., 1985).

1.4 ARTHROPOD VECTORS OF BEFV

To date, epidemiological evidence strongly suggests that BEFV is transmitted by a haematophagus arthropod vector (Blackburn et al., 1985; Cybinski et al., 1990;

Davies and Walker et al., 1974; Lvov et al., 2015; Muller and Standfast, 1986; St

George et al., 1976). In a number of studies performed in Africa (Blackburn et al.,

1985; Davies and Walker et al., 1974), Australia (Cybinski et al., 1990), and Eurasia

(Lvov et al., 2015), BEFV was isolated from biting midges belonging to the genus

Culicoides spp. Despite these reports, however, biological transmission of BEFV by biting midges has never been demonstrated (Walker and Klement, 2015). Similarly,

BEFV was isolated from different species of mosquitoes belonging to different genera including Anopheles bancrofti and a mixed pool of Culex spp, Aedes spp, and Uranotaenia spp (Muller and Standfast, 1986; St George et al., 1976). While replication of BEFV is believed to occur in the arthropod vector, however, vector competence has not been definitively demonstrated in both midges and mosquitoes

(Walker and Klement, 2015). Since BEF outbreaks tend to occur immediately after the onset of heavy rains in geographical areas that include those that are free from

Culicoides spp (Kirkland et al., 1993), however, it has been concluded that mosquitoes, and not midges, are the most likely major biological vectors of the virus.

In a Kenyan study performed by Davies et al. (1990), however, the distribution of

BEFV was reported to extend beyond the zones in which mosquitoes are abundant, and its appearance in locations from which other mosquito-borne diseases like Rift

7 valley fever have not been isolated suggests that transmission by midges is also possible.

1.5 PATHOGENESIS OF BEF

1.5.1 Clinical and pathological features

In some but not all BEFV-infected cattle, the disease is characterised by transient polyphasic fever, inappetance, synovitis, muscle stiffness, lameness, and paresis and/or paralysis (Basson et al., 1970; St George et al., 1995; Young and Spradbrow,

1990a). In addition, clinically sick animals also often exhibit biochemical blood abnormalities including but not limited to hypocalcaemia and hyperfibrinogenaemia

(St George et al., 1984; Young and Spradbrow, 1990a). In studies on serum biochemical dyscrasias undertaken in Australia, hypocalcaemia was reported to be a feature of acute BEF (St George et al., 1984; St George et al., 1995; Uren et al.,

1992) even when the precise mechanism was not investigated. In a groundbreaking study reported by Nielsen et al., (1997), high doses of IL-1β were linked to upregulated transcription of the parathyroid CaSR gene in bovine parathyroid tissue slices (Nielsen et al., 1997). As the CaSR molecule is an important regulatory element in the complex mechanism of blood calcium homeostasis, it may be speculated that increased expression of the molecule as a result of high levels of circulating proinflammatory cytokines may, in part, lead to hypocalcaemia in acute

BEF. Besides, IL-6, which is another important proinflammatory cytokine, was shown to suppress the secretion of parathyroid hormone (PTH) by bovine parathyroid cells cultured in vitro (Carstedt et al.,1999) further underscoring the role proinflammatory cytokines may play in disturbing the homeostasis of blood calcium. Future in vivo studies in cattle are required to further assess the validity of these hypotheses.

While several studies have described the clinical and pathological aspects of BEF in cattle (Basson et al., 1970; Young and Spradbrow, 1980a; Young and Spradbrow,

1990b), major gaps still remain in our understanding of the pathogenesis of the disease. A number of studies have indicated that inflammation and fever are hallmark features of acute BEF (Basson et al., 1970; Young and Spradbrow, 1990a).

Pathological changes that allude to the inflammatory nature of this disease include but are not limited to neutrophilia, polyserositis, vasculitis, tenovaginitis, and fibrinous exudates within the pleural, peritoneal, pericardial and joint cavities (Basson et al.,

1970). While it is possible that pro-inflammatory cytokines may initiate the inflammatory lesions (St George et al., 1995; Uren et al., 1989), the precise mechanisms that underlie polyserositis and related lesions are poorly understood. In a study reported by Young (1990), increase in vascular permability particulary involving blood vessels associated with serosal surfaces was demonstrated by the use of colloidal carbon or Evans blue dye. This author speculated that immune complex deposition and subsequent inflammation probably contributed to vasculitis and dependant lesions (Young, 1990). Other than this report, little has been done to elucidate the mechanisms of inflammation and increased vascular permeability seen in BEF.

1.5.2 Role of cytokines in the pathogenesis of BEF

In the in vitro study reported by Chang et al. (2004), BEFV was able to induce caspase-dependent apoptosis in cultured BHK cell lines (Chang et al., 2004).

Subsequent to this study, Lin et al. (2009) showed that BEFV-induced in vitro apoptosis requires viral gene expression and is associated with activation of Fas and mitochondrion-mediated caspase-dependent pathways (Lin et al., 2009). Despite the

9 few in vitro studies in which apoptosis has been demonstrated in BEFV-infected BHK cell lines (Chang et al., 2004; Lin et al., 2009), definitive evidence of an in vivo cytolytic activity of BEFV has not been adduced (Young and Spradbrow, 1990b;

Uren et al., 1992). As such, some researchers have speculated that proinflammatory cytokines may play a pivotal role in the pathogenesis and clinical expression of acute

BEF in cattle (St George et al., 1995; Uren et al., 1989; Uren et al., 1992). This hypothesis was further supported by the resolution of fever following treatment of

BEFV-infected cattle with the anti-inflammatory drug phenylbutazone (Uren et al.,

1989). In a conference paper presented by Uren and Zakrzewski (1989), increased expression of plasma IL-1β and TNF-α was seen in febrile cattle that had been experimentally infected with BEFV. While this report suggests a potential role of proinflammatory cytokines in the pathogenesis of BEF (Uren and Zakrzewski, 1989), conclusive studies have never been done to corroborate the hypothesis in naturally infected adult cattle.

1.5.3 Tissue tropism of BEFV

Since the pioneering BEF studies in Australia (Burgess and Spradbrow, 1977;

Mackerras et al., 1940; St George, 1986; Young and Spradbrow, 1990a) and South

Africa (Basson et al., 1970), the in vivo replication sites and the fate of the BEFV after the viraemic phase have not been empirically defined in cattle. In studies based on fluorescent antibody tests, intracellular viral antigens were demonstrated in tissues and within cellular elements derived from serosal fluids taken from BEFV- infected cattle (Odiawo et al., 1992; Theodoridis, 1969; Young and Spradbrow,

1990). In one of these studies, viral antigens appeared to be located intracytoplasmically within neutrophils as well as within reticular cells in the spleen

10 and lymph node (Young and Spradbrow, 1990b). While these studies were inconclusive, the research findings led the authors to postulate that reticuloendothelial tissues were probable replication sites of the BEFV (Young and

Spradbrow, 1990b). Besides, while a number of studies have shown the virus to be associated with circulating neutrophils during the viraemic phase of the disease

(Theodoridis, 1969; Young and Spradbrow, 1980; Young and Spradbrow, 1990b), it is not clear if this is simply an outcome of neutrophil-mediated phagocytosis rather than the inherent ability of the virus to actively invade and replicate inside these cells. In a symposium report authored by Odiawo et al., (1992), BEFV was isolated from the bone marrow of three experimentally-infected cattle before the onset of detectable viraemia. Based on this observation, the authors proposed that the bone marrow was a potential site of virus replication even when a specific host cell type that could support such replication was never defined.

1.5.4 Potential neurotropism of BEFV

Of the several biochemical abnormalities detectable in the blood during acute BEF in cattle, hypocalcaemia may arguably contribute to neuromuscular weakness and paresis or paralysis (St George et al., 1986). As such, the hypocalcaemia-induced paresis/paralysis is easily reversed by parenteral infusion of calcium borogluconate

(St George et al., 1986; Nandi and Negi, 1999). However, the basis of chronic paralysis seen in some BEFV-infectedinfected but otherwise normocalcaemic cattle

(St George et al., 1986) is not clear. In an Australian study reported by Tzipori

(1975), fatal encephalitis was induced by intracerebral inoculation of a virulent BEFV strain into two seronegative calves but not an age-matched animal that was seropositive to maternal virus neutralising antibodies. Subsequent to that finding,

11 severe bilateral and symmetrical Wallerian degeneration (WD) was also demonstrated in spinal cord sections of four BEFV-infected animals (Hill and Shultz,

1977), based on which these authors speculated that compressive myelopathy resulting from probable mechanical trauma was the likely cause of the lesions and chronic paralysis in the affected animals. In three of the four animals, the WD was present in the first segment of the cervical spinal cord while in the other, it was localised to the lumbar spinal cord. While these findings have provided evidence of the basis of some of the neurological symptoms seen in a number of BEFV-infected cattle, the mechanistic events that underlie paresis and chronic paralysis in some field BEF cases that do not show spinal cord lesions remains a subject of scientific speculation.

1.6 MECHANISMS OF IMMUNITY IN BEF

1.6.1 Humoral immunity

Both natural BEFV infections and vaccination with BEFV-derived immunogens are typically followed by a long-lived solid immunity that is attributed to virus neutralising antibodies (Aziz-Boaron, 2013; Mackerras et al., 1940; Uren et al., 1994). While humoral immunity based on virus neutralising antibodies is quite protective (Aziz-

Boaron et al., 2013; Uren et al., 1994), there have been cases of experimentally infected and naturally exposed cattle with positive virus neutralising antibody titres that succumb to experimental challenge and/or even natural BEFV infections (Aziz-

Boaron et al., 2014; Della-Porta and Snowdon, 1979; Tzipori and Spradbrow, 1973).

Evidence available to date indicates that BEFV strains collected from different geographical areas of the world may be grouped under one serotype (Davies and

Walker, 1974; Inaba et al., 1969; Snowdon, 1970; Kemp et al., 1973; Tian et al.,

1987; Walker and Klement, 2015). In the work reported by these authors, cross-

12 reactions were documented between BEFV strains originating from Australia, Asia, and Africa (Davies and Walker, 1974; Inaba et al., 1969; Kemp et al., 1973;

Snowdon, 1970; Tian et al., 1987; Walker and Klement, 2015). In another in vitro study, a mAb developed against a specific site within one of the BEFV envelope glycoproteins neutralised different strains of BEFV plus BRMV and KIMV (Cybinski et al., 1992). In addition, there is evidence that the neutralising antibody response that develops during KIMV infection in cattle may be protective against BEFV infection (Cybinski, 1987). Of note, phylogenetic studies of different BEFV strains from diverse geographical locations have since provided insights on the likely molecular basis of such cross-reactions (Kato et al., 2009; Ting et al., 2014). For example, nucleotide and amino acid sequence similarities higher than 96% were documented in BEFV strains originating from Taiwan, China and Japan (Ting et al.,

2014). Such a high degree of genetic similarities amongst different BEFV strains from different geographical locations is a great advantage as vaccines developed against a specific BEFV strain are potentially cross-protective for cattle against heterologous strains from geographically diverse regions of the world (Walker and

Klement, 2015). While this is the case, however, homologous BEFV strains confer far superior immunoprotection in cattle (Inaba et al., 1969; Tian et al., 1987; Walker and Klement, 2015).

Of the various members of the Family Rhabdoviridae, relatively more studies on humoral immunity have been performed on the rabies virus in domestic animals and humans (Chatchen et al., 2017; Harvey et al., 2016). In a study of rabies immunity in horses, protective neutralising antibody titres were maintained for a period of 2 to 3 years in animals that had been vaccinated with a rabies immunogen (Harvey et al.,

2016). Likewise, rabies vaccination in Thai children induced protective virus neutralising antibody titres that lasted up to three years with sub-protective titres lasting up to 8 years (Chatchen et al., 2017). Regarding BEF in cattle, the virus neutralising antibody response seen in natural BEFV infection and the immunoprotection associated with it are prolonged and believed to last several years

(Aziz-Boaron et al., 2013; Uren et al., 1994).

1.6.2 Cell-mediated immunity

While post-infection/vaccination immunity in BEF has been largely attributed to virus neutralising antibodies (Uren et al., 1994; Aziz Boaron et al., 2013), the contributory role of cellular immune mechanisms in the development of immunity against BEFV has not been exhaustively investigated. In particular, the role(s) played by T lymphocytes and specific cytokines in driving the innate-adaptive immune response transition have not been investigated in cattle infected with the virus. However, despite these knowledge gaps, some authors have suggested, albeit without definitive evidence, that cell-mediated immune mechanisms may contribute to post- infection immunity in BEF (Della-Porta and Snowdon, 1979; Uren et al., 1993; Uren and Zakrzewski, 1989). In a conference report authored by Uren et al. (1993), T-cell lines established from peripheral blood mononuclear cells (PBMCs) derived from cattle immunised with a subunit immunogen proliferated in vitro when exposed to native BEFV antigens. Interestingly, IL-2, a T-cell derived cytokine that is mitogenic for T cells, was demonstrated in supernatants of T-cell cultures (Uren et al., 1993).

This finding suggests the possibility of cellular mechanisms playing a role in the development of adaptive immunity against BEFV. While these data provided strong evidence to build a hypothesis on adaptive cell-mediated immunity in BEF, in vivo

14 studies have to date not been done to corroborate these findings. Moreover, the cellular mechanisms that prevail during the innate-adaptive immune response transition period have not been studied. Similarly, the role of T-helper cells in mounting successful antibody responses at the time of seroconversion in BEFV- infected cattle also remains unstudied.

1.7 CONCLUDING REMARKS

While BEF is of great economic and animal welfare significance, the pathogenesis of the disease is poorly understood. As such, there are still gaps in developing highly efficacious vaccines and designing effective treatments for BEF. While some unpublished work done in Australia has suggested the potential role of proinflammatory cytokines in the pathogenesis of fever and related symptoms in BEF, empirical studies based on naturally infected cattle have not been done to corroborate these reports. As proposed in the present study, describing the precise mechanisms that underlie inflammation in BEFV-infected cattle has the potential to inform future researches into the development of novel BEF treatments. Post- infection/vaccination immunity in cattle has mostly been attributed to virus neutralising antibodies (Aziz-Boaron et al., 2013; Uren et al., 1994) without delineating the contributory role(s) played by cellular immune mechanisms. For example, the role played by Th1 and Th2 cytokines in mediating anti-BEFV immune mechanisms has not been investigated, yet any successful design of efficacious BEF vaccines requires such knowledge. In particular, the role(s) played by cytokines in driving the innate-adaptive immune response transition during BEF remain unstudied thus justifying the present study. Considering the widely accepted concepts on mammalian immunobiology, it is plausible that adaptive immunity in BEF is likely to

15 require such cellular mechanisms for potentiating the virus neutralising antibody response. In a conference report by Uren et al, (1993), T-cell lines established in vitro from peripheral blood mononuclear cells collected from cattle immunised with a subunit BEFV immunogen proliferated in presence of native BEFV antigens. In these studies, increased IL-2 levels were demonstrated in supernatants of T-cell cultures suggesting possible cellular events involved in BEF immunity. While these findings clearly provide anecdotal evidence for a possible role of T-cell immune mechanisms against BEFV (Uren et al., 1993), however, in vivo cellular mechanisms in BEF in cattle remain unstudied. The present study was therefore designed to generate new knowledge that will potentially pave way to more elaborate T-cell studies which once conducted will inform research into highly efficacious anti-BEF vaccines.

It should also be noted that despite studies spanning several decades, little is known about the tissue tropism of BEFV in cattle. Intracellular viral antigens were previously demonstrated by the fluorescent antibody test in tissue specimens and within cellular components of serosal fluids taken from BEFV-infected cattle (Burgess and

Spradbrow, 1977; Theodoritis, 1969; Young and Spradbrow, 1985). In one of these studies, the intracytoplasmic viral antigens appeared to be located within neutrophils as well as reticular cells in the spleen and lymph node (Burgess and Spradbrow,

1977). Based on these findings, this research was premised on the hypothesis that tissues of the reticuloendothelial system are potential replication sites for the BEFV.

Describing the potential tissue replication sites of BEFV will not only advance our knowledge on the pathogenesis of BEF but will also improve BEF diagnostics by elucidating on the best biological samples to test for during diagnostic investigations of cattle dying from suspected BEF.

Of the several biochemical abnormalities detectable during acute BEF, it is now known that hypocalcaemia may contribute to neuromuscular weakness and therefore paresis/paralysis (St George, 1986). However, this does not seem to account for the paralysis seen in some BEFV-infected but otherwise normocalcaemic cattle (St

George, 1986). The hypocalcaemia-induced paresis/paralysis is easily reversed by parenteral infusion of calcium borogluconate (Nadi and Negi, 1999; St George et al.,

1984). In the mid-1970s, fatal encephalitis was experimentally induced by intracerebral inoculation of a virulent BEFV strain into two seronegative calves but not an age-matched animal that was seropositive for maternal virus neutralising antibodies (Tzipori, 1975). Subsequently, severe bilaterally symmetrical Wallerian degeneration (WD) was demonstrated in spinal cord sections of four BEFV-infected animals prompting the authors to speculate that compressive myelopathy was the likely cause of these lesions (Hill and Schulz, 1977). In three of the four animals, the

WD was present in the first segment of the cervical spinal cord while in the other, it was localised in the lumbar spinal cord. By elucidating the basis of pares/paralysis in some of the BEFV-infected animals, this study will not only advance our knowledge on the pathogenesis of the neurological aspects of BEF but also inform the design of novel and more effective treatment protocols for the disease.

1.8 AIMS OF THIS STUDY

1. To describe the plasma kinetics of IL-1β, IL-6, IL-10, and TNF-α during acute

BEF in order to assess how cytokine expression is temporally associated with

fever in adult cattle naturally infected with BEFV.

2. To describe the kinetics of Th1-related cytokines (IL-2 and IFN-γ), IL-6, and

IL-10 during the innate immune response transition in adult cattle infected with

BEFV as a way of elucidating the potential contributory role of cell-mediated

mechanisms in the development of immunity against natural BEFV infections

in cattle.

3. To evaluate the relationship between the kinetics of the virus neutralising

antibody response and the resolution and duration of viraemia.

4. To determine the tissue tropism of BEFV and elucidate the potential in vivo

tissue replication sites of the virus in cattle.

5. To assess the potential neurotropism of BEFV in naturally infected cattle, and

describe the different histomorphological abnormalities in the central and

peripheral nervous tissue that may be causally linked to chronic paralysis

observed in few BEFV-infected animals.

CHAPTER 2

KINETICS OF THE VIRUS NEUTRALISING ANTIBODY RESPONSE AND

SELECTED PLASMA CYTOKINES DURING ACUTE DISEASE AND THE

INNATE-ADAPTIVE IMMUNE RESPONSE TRANSITION IN CATTLE NATURALLY

INFECTED WITH THE BOVINE EPHEMERAL FEVER VIRUS

This chapter is a composite of data published as two research articles. For consistency of formatting within the thesis and to minimise self-plagiarism concerns, extensive modifications have been made to the already published text.

1. Research paper published in the Journal of Veterinary Microbiology:

Barigye, R.,1,2,3 Melville, L.F.,1 Davis, S.,1 Walsh, S.,1 Hunt, N.,1 Hunt, R.1. (2016).

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus. Vet Microbiol

186: 111–116

2. Research paper published in the Journal of Veterinary Immunology and

Immunopathology:

Barigye, R.,1,2,3 Melville, L.F.,1 Davis S.,1 Walsh, S.,1 Hunt, N.,1 Hunt, R.,1 Elliott, N.1.

(2015). Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle. Vet

Immunol and Immunopathol 168:159–163.

1Berrimah Veterinary Laboratories, Department of Primary Industry and Resources,

GPO Box 3000, Darwin, NT 0801, Australia;

2School of Veterinary Science, University of Queensland, Gatton QLD 4343,

Australia;

3Department of Veterinary Medicine, College of Food and Agriculture, United Arab

Emirates University, PO Box 15551, Al Ain, UAE

2.1 ABSTRACT

While fever and inflammation are hallmark features of BEF, the kinetics of cytokine expression during the acute phase of the disease have not been empirically defined in adult cattle naturally infected with the BEF virus (BEFV). Furthermore, while virus neutralising antibodies are variably protective against BEFV infections, the cytokine events that mediate the nascent adaptive immune response have not been defined.

The first set of studies characterised the plasma kinetics of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and IL-10 during acute BEF as well as elucidated the relationship between the onset of the virus neutralising antibody response and cessation of viraemia in adult cattle naturally infected with BEFV. In the second set of studies, the kinetics of plasma IL-2, IFN-γ, IL-6, and IL-10 were evaluated during the period of innate immune response transition and the relationship between the virus neutralising antibody response and viraemia was also assessed. Plasma samples from three BEFV-infected and three uninfected cattle from the first set of studies were tested for IL-1β, IL-6, TNF-α, and IL-10 using cELISA, viraemia was monitored by qRT-PCR, and virus neutralising antibody titres determined using a standard protocol. Clinical monitoring, including measurement of rectal temperatures, was also done on all the study cattle. In relation to the second cohort of cattle, plasma samples taken from four virus-infected and uninfected negative control animals were tested for IL-2 and IFN-γ (‘Th1-indicator cytokines’) and IL-6

20 and IL-10 (‘Th2-indicator cytokines’) by cytokine-specific ELISAs, and viraemia and virus neutralising antibody titres monitored by qRT-PCR and a standard protocol, respectively. In the first cohort of cattle, plasma concentrations of IL-1β, TNF-α, IL-6, and IL-10 were consistently higher than in the three virus-infected animals but not in the negative controls. Two of the infected animals were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already underway by the time viraemia was detected in all the three infected heifers. In all the BEFV- positive animals, IL-1β was the most strongly expressed cytokine, while plasma IL-6 and IL-10 showed intermediate levels, and TNF-α demonstrated the least plasma concentrations. In addition, TNF-α demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. In respect to the second cohort of cattle, plasma IL-6 and IL-10 levels were higher in all the virus-infected but not the negative controls, and the onset of the cytokine response started several days prior to the initiation of viraemia. In one heifer, plasma IL-2 and IFN-γ levels were consistently higher than in the other three virus-infected animals and the negative control. Notably, the heifer with the highest IL-2 and IFN-γ response had the shortest viraemia while the animal with the lowest IL-2/IFN-γ indices showed the longest viraemia. The data from the first cohort of cattle document variable increase in plasma IL-1β, IL-6, TNF-α, and IL-10 during natural BEFV infection and further indicate that upregulation of all but TNF-α precedes seroconversion. Based on these data, it is likely that cytokine-mediated cellular mechanisms may be required to augment virus neutralising antibodies as a requirement for the resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may

21 potentially antagonise fever response in BEFV-infected cattle. As suggested by data from the second cohort of cattle, the increase in plasma IL-6 and IL-10 appears to precede seroconversion during BEFV infections suggesting that the two cytokines may influence immunological events, such as those that pave way to B-cell activation and seroconversion. While remarkable variability of IL-2 and IFN-γ expression was observed in the BEFV-infected animals from the second cohort, increased plasma levels of the two cytokines appear to be associated with a shorter viraemia. Detailed cellular immunology studies are needed to help define the precise role of T cells in the anti-BEFV adaptive immune responses.

2.2 INTRODUCTION

As there is no evidence that BEFV is cytolytic (Young and Spradbrow, 1990b; Uren et al., 1992), the effect of proinflammatory cytokines is believed to play a significant role in the pathogenesis and clinical expression of acute BEF in cattle (St George et al., 1995; Uren et al., 1989; Uren et al., 1992). In a study carried out in Australia, treatment of experimentally BEFV-infected cattle with the anti-inflammatory drug phenylbutazone was able to eliminate the fever response (Uren et al., 1989). In a conference paper reported by Uren and Zakrzewski (1989), increased expression of plasma IL-1β and TNF-α was seen in febrile cattle that had been experimentally infected with BEFV. While this report alludes to the potential role of proinflammatory cytokines in the pathogenesis of BEF (Uren and Zakrzewski, 1989), conclusive studies have not been done in naturally infected adult cattle to further corroborate this hypothesis.

In regard to the immunity developed against BEFV in cattle, both natural BEFV infections and vaccination with BEFV-derived immunogens are most of the time followed by solid immunity that, thus far, has been attributed to production of virus neutralising antibodies (Uren et al., 1994; Aziz-Boaron et al., 2013). However, in a number of studies, several animals vaccinated with an inactivated BEFV-derived vaccine also succumbed to challenge BEFV infection despite having attained high neutralising antibody titres (Aziz-Boaron et al., 2014; Della-Porta and Snowdon,

1979; Tzipori and Spradbrow, 1973). Therefore, it is most likely that adaptive immunity in BEF also involves cellular mechanisms that arguably augment the virus neutralising antibody response in promoting virus clearance and immunity. While this is likely to be true, however, such a contributory role of cell-mediated immune mechanisms in the development of the anti-BEFV immunity is still poorly understood.

In a conference report by Uren et al. (1993), T-cell lines established in vitro from peripheral blood mononuclear cells (PBMCs) taken from cattle that had been immunised with a subunit BEFV immunogen proliferated in presence of native BEFV antigens. Interestingly, increased IL-2 levels were demonstrated in supernatants of the T-cell cultures suggesting that the presumed cellular events may precede seroconversion in BEF. While these findings provide anecdotal evidence of a possible role of T-cells in anti-BEFV immune responses (Uren et al., 1993), however, the presumed in vivo cellular mechanisms that characterise the nascent adaptive immune response in cattle with BEFV infections remain largely unstudied.

The study based on the first cohort of cattle was conducted to describe the plasma kinetics of IL-1β, IL-6, IL-10, and TNF-α during acute BEF as well as to assess whether increased cytokine production is temporally associated with fever in adult

23 cattle naturally infected with BEFV. In this study, the relationship between the onset of virus neutralising antibodies and resolution of viraemia was also evaluated. In the study based on the second cohort of cattle, the kinetics of IL-2 and IFN-γ (Th1 indicator cytokines’), as well as IL-6, and IL-10 (‘Th2 indicator cytokines’) during the innate-immune response transition in adult cattle naturally infected with BEFV were described. In addition, the relationship between the virus neutralising antibody response and the duration of viraemia was evaluated.

2.3 MATERIALS AND METHODS

2.3.1 Study animals

These experiments were conducted during natural BEF outbreaks and included 14 adult Brahman cattle selected from two sentinel herds totalling 42 animals (cohort

No. 1 = 6/24 cattle; cohort No. 2 = 8/18 cattle). During these studies, the animals were humanely treated according to the ethical guidelines stipulated in the Animal

Ethics Committee Approval No. A12036. For the study based on the first cohort of animals, the six-day clinical monitoring period included recording of rectal temperatures, observation of BEF-characteristic clinical signs, as well as blood sample collections that were initiated as soon as the index case of BEF was confirmed by a positive qRT-PCR result. The 24 sentinel cattle were studied during a

BEF outbreak at the Berrimah Research Farm in Darwin, NT. Blood samples collected during the BEF outbreak infected were tested for virus growth by the virus isolation test. To evaluate the presence of BEFV and the other regionally prevalent arboviruses of animal health significance including Akabane, blue-tongue, and epizootic haemorrhagic viruses, the blood samples were screened for virus neutralising antibodies by means of virus neutralisation tests. In addition, qRT-PCR

24 was used to detect BEFV viral RNA, and plasma samples taken from the study cattle were tested for plasma cytokines including IL1- β, IL-6, TNF- α, and IL-10. For the first cohort, the animals whose samples tested positive for BEFV by qRT-PCR and/or virus neutralising antibodies but were negative for viral growth, and/or seronegative to the other regionally prevalent arboviruses of animal health significance including

Akabane, blue-tongue, and epizootic haemorrhagic viruses met the inclusion criteria for the study (n=3). Samples negative for BEFV by qRT-PCR and/or BEFV neutralising antibodies as well as those that were negative for viral growth and/or seronegative for the other regionally prevalent arboviruses of animal health significance met the inclusion criteria of negative controls (n=3). On the other hand, the blood samples from the remaining 18 animals either resulted in virus growth and/or were found seropositive to virus neutralising antibodies for at least one of these viruses and as such were excluded from the study. For the study based on the second cohort of cattle (N=18 cattle), the nine-day clinical monitoring period included recording of rectal temperatures, observations of BEF characteristic clinical signs, as well as blood sample collections initiated as soon as the index case of BEF was confirmed by a positive qRT-PCR result. The sentinel cattle were studied during a

BEF outbreak at the Beatrice Hill Research Farm, Northern Territory located about

60 km from Darwin. The collected blood samples were tested for virus growth by virus isolation and by the virus neutralisation test for virus neutralising antibodies to

BEFV and the other regionally prevalent arboviruses of animal health significance. qRT-PCR was also used to detect BEFV viral RNA. Those samples that were found positive for BEFV by qRT-PCR and/or virus neutralising antibodies but were negative for viral growth and/or seronegative to the other regionally prevalent arboviruses of animal health significance met the inclusion criteria as BEFV-infected study subjects

(n=4). On the other hand, the samples that were found negative for BEFV by qRT-

PCR and/or BEFV neutralising antibodies as well as those that were negative for viral growth and/or seronegative by virus isolation and virus neutralisation antibody test respectively to the other regionally prevalent arboviruses of animal health significance met the inclusion criteria as the negative controls (n=4). The blood samples from the remaining 10 animals were excluded from the study because they were either positive for virus growth and/or were found seropositive to virus neutralising antibodies for at least one of the other regionally important arboviruses as listed above. During the experiments, all the animals were humanely treated according to ethical guidelines stipulated in the Animal Ethics Committee Approval

No. A12036. Plasma samples collected during the monitoring period were tested for plasma cytokines including IL-2, IFN-γ, IL-6 and IL-10.

2.3.2 qRT-PCR for BEFV

The qRT-PCR protocol used for BEFV detection during these studies was previously standardised by Lew et al. (2006) with minor in-house modifications performed at the

Berrimah Veterinary Laboratories. Viral RNA was extracted using the MagMAX-96 viral RNA isolation kit (Applied Biosystems, CA, USA) from blood samples collected in EDTA tubes, eluted with 50 µl elution buffer, and stored at −80◦C until testing. The qRT-PCR assay was optimised with the AgPath-ID One-Step RT-PCR Kit (Applied

Biosystems, CA, U.S.A.) using a total volume of 25 µl. Briefly, a master mix consisting of 4.0 µl nuclease-free water, 12.5 µl 2× RT-PCR buffer, 1.0 µl 25 × RT-

PCR enzyme mix,1.0 µl BEFVgF2 forward primer (10 µM), 1.0 µl BEFVgR2 reverse primer (10 µM) and 0.5 µl BEFVgMGB (TaqMan®) Probe (5µM) for one reaction was prepared and 5 µl RNA template was added (The primers and concentrations used

26 in this study are indicated in Table 2.1 below.). For amplification, the following temperature profile was used: 30 min at 50◦C (reverse transcription), 10 min at 95◦C

(inactivation reverse transcriptase/activation Taq polymerase), followed by 50 cycles of 15 s at 95◦C (denaturation), 60 s at 60◦C (annealing and elongation). A positive and a negative extraction control, and positive and negative reverse transcription and amplification controls were included in the assay to verify the correctness of the nucleic acid extraction and/or the qRT-PCR reaction. The details regarding the different controls used in the qRT-PCR assay may be found in Appendix 3.

Table 2.1: Primer probes used in the qRT-PCR assay for the detection of BEFV RNA in the present study (Lew et al., 2006).

Name Sequence 5’→3’ Final Conc

BEFVgF2 TTT TAT CWG CTG TTG TAG GTT GGT 800 nM

BEFVgR2 AAC AGC CCA AAT TGT CCA TCT T 800 nM

BEFVgMGB (TaqMan® Probe) 6FAM-ACG GCA AAG GCA G-MGBNFG 100 nM

2.3.3 Virus neutralisation test

The modified version of the virus neutralisation test (Uren et al., 1994) applied in the present study involved testing of preheated serum samples in 96-well plates containing Minimum Essential Medium or MEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal bovine serum. To conduct the assay, 50 µl of serially diluted pre-heated sera were held in quadruplicate wells and incubated with 50 µl of

MEM containing 100 TCID50 of BEFV for 1 h at 37◦C. Then, 100 µl of MEM containing BSR cells at a concentration of 2 × 105 cells per ml was added, and the

27 plates were incubated at 37◦C and then read for cytopathic effect after five days

(Sato et al., 1975). The virus neutralising antibody titres were calculated using the

50% end point method originally reported by Reed and Muench (1938).

2.3.4 Immunoenzymatic assays for bovine cytokines

For the first cohort of cattle, the cytokine (IL-1β, IL-6, IL-10, and TNF-α) assays were performed using Cusabio® competitive inhibition ELISA (cELISA) according to the manufacturer’s instructions and the plates read at 450 nm. Using the “Curve Expert

1.3” software program (Hyams Development, AL, USA), the protein standards OD450 readings were used to generate a standard curve which was then used to derive the plasma cytokine concentrations as specified in the Cusabio® cELISA kit instructions.

For the second cohort of cattle, the plasma samples collected from three days prior to the detection of viraemia through to the ninth day of the study were tested for IL-2, and IFN-γ (‘Th1 indicator cytokines’) as well as IL-6 and IL-10. IL-6 and IL-10 were tested using the cytokine-specific Cusabio® cELISA according to the manufacturer’s instructions. Using the “Curve Expert 1.3” software program (Hyams Development,

AL, USA), the OD450 readings for the protein standards were plotted and a standard curve generated. The plasma concentrations of IL-6 and IL-10 in the test samples were then derived by extrapolating the respective OD450 readings onto the standard curve. In the case of IL-2 and IFN-γ, plasma samples were tested by the Cusabio® antigen capture ELISA according to the manufacturer’s instructions. SinceIL-2 and

IFN-γ concentrations in some of the samples were slightly out of the detection limit of the standard curve, plasma levels of the two cytokines were inferred from the magnitude of the OD450nm readings.

2.4 RESULTS

2.4.1 Study cattle – Cohort 1

2.4.1.1 Fever, seroconversion, and resolution of viraemia

On day 1 of clinical monitoring, the BEFV-infected animals No. B113 and B118 were clinically ill, recumbent, and had rectal temperatures (recT) of 39.1ºC and 41.0◦C, respectively (Table 2.2). However, the fever subsided by day 2 of clinical monitoring.

The other focus of this study was to evaluate whether neutralising antibodies were the only important factor in the resolution of viraemia during acute to subacute BEF.

As may be noted, viraemia was observed for two days in the two febrile heifers and promptly resolved when the neutralising antibody titres reached 8 and 16, respectively (Table 2.3). In the other virus-infected animal, however, viraemia continued for three days after seroconversion and only subsided when the antibody titre reached 72 (Table 2.3).

Table 2.2: Rectal temperatures (◦C) of the six study cattle during the six-day monitoring period.

Days

1 2 3 4 5 6

Bovine ephemeral fever infected (n=3)

B113 39.1* 38.6 38.9 38.7 38.8 38.5

B118 41.0* 38.2 38.1 38.4 37.9 38.4

B121 38.7 38.6 39.3* 38.7 38.2 38.5

Uninfected negative controls (n=3)

B101 37.8 38.5 38.0 37.7 38.7 38.3

B114 38.6 39.0 38.5 38.5 38.6 38.6

B115 38.1 38.5 38.3 38.3 38.5 38.5

Legend: Febrile temperatures are highlighted in bold fonts along with an asterisk.

Rectal temperatures ≥39.0 were considered to be consistent with a febrile response.

Table 2.3: Virus neutralising antibody titres and qRT-PCR data for the three cattle naturally infected with the bovine ephemeral fever virus.

Days

1 2 3 4 5 6 7 8

Bovine ephemeral fever virus infected (n=3)

B113 <4 <4 8 32 10 18 6 6

(20.3) (24.5) (>45) (>45) (>45) (>45) (>45) (>45)

B118 <4 <4 16 48 96 144 160 160

(18.7) (20.6) (>45) (>45) (>45) (>45) (>45) (>45)

B121 <4 <4 <4 40 64 64 36 72

(25.3) (18.6) (18.7) (21.0) (29.6) (>45) (36.6) (>45)

Legend: The bold numerals represent the virus neutralising antibody titres while the numbers in parentheses are the Ct values obtained from the qRT-PCR assay. The boxes shaded in grey denote days when peripheral blood was positive for viral RNA when tested by qRT-PCR. Note that all the three negative controls had negative virus neutralising antibody titres of <4 and negative Ct values (qRT-PCR) of ≥45.

2.4.1.2 Kinetics of plasma cytokines (IL1- β, IL-6, TNF- α, and IL-10)

Of the four cytokines studied in the first cohort of cattle, IL-1β attained the highest plasma concentrations in the three virus-infected heifers in which picogram values of the proinflammatory cytokine were consistently greater than those of IL-6, TNF-α, and IL-10 (Figure 2.1). When plasma IL-1β kinetics were further evaluated, triphasic peaks were apparent in the virus-infected heifers around days 1, 4 and 6.

Interestingly, the highest IL-1β peak occurred around day 4, which was approximately the time IL-6, and IL-10 concentrations were in equilibrium (Figure

2.1; Figure 2.2D). Moreover, a visual appraisal of the data graphics demonstrated a somewhat similar pattern between the mean recT and mean plasma IL-1β concentrations for the BEFV-infected cattle (Figure 2.2A) but not for TNF-α and IL-6

(Figure 2.2B and 2.2C).

Figure 2.1: Comparative kinetics of plasma cytokines (IL-1β, IL-6, IL-10, and TNF-α) in three adult cattle naturally infected with bovine ephemeral fever virus and three uninfected age-matched negative controls.

Figure 2.2: Relationship between the mean rectal temperature and mean plasma concentrations of IL-1β, TNF-α and IL-6 (A–C) in adult cattle naturally infected with the bovine ephemeral fever virus (n = 3). The pre-seroconversion plasma kinetics of

IL-6 and IL-10 are shown in Fig. 2D. The bold arrow indicates the first day viraemia was confirmed by a positive BEFV-qRT-PCR result

2.4.2 Study cattle – Cohort 2

2.4.2.1 Viraemia and kinetics of the virus neutralising antibody response

For all the four cattle infected with BEFV, viraemia was first detected on day 4 and lasted only three days in animal No. B20, four days in B05 and B10, and six days in

B11 (Table 2.4). With the exception of animal B11, the viraemia in B05, B10 and B20 resolved one day after seroconversion when the neutralising antibody titres were 64,

144 and 72, respectively. In animal B11, on the other hand, the viraemia continued for six days and only subsided after the neutralising antibody titre rose to 144 (Table

2.4). In B05, following cessation of viraemia, the antibody response continued to rise, peaked around day 13, before starting to decline gradually until day 15 when the observations were concluded. In animal B10, the antibody response peaked on day

11 and thereafter started declining albeit with some minor antibody titre fluctuations.

In B11, the antibody response peaked on day 12 and thereafter also started declining. In animal B20, on the other hand, the antibody response peaked on day 8 before showing a gradual decline that was associated with characteristic daily fluctuations.

Table 2.4: The relationship between virus neutralising antibody response and viraemia during natural bovine ephemeral fever infections in four adult Brahman cattle.

Day from study onset 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Bovine ephemeral fever virus infected group (n=4)

Animal ID No. (Virus neutralising antibody titres and qRT-PCR Ct values)

B05 <4 <4 <4 <4 (23.2) <4 (19.4) <4 (17.7) 4.5 (20.1) 64 144 160 160 192 288 256 256

B10 <4 <4 <4 <4 (27.6) <4 (19.1) <4 (19.2) 4.5 (28.6) 144 64 640 768 144 160 144 128

B11 <4 <4 <4 <4 (19.1) <4 (36.9) <4 (29.2) <4 (27.9) 4.5 (25.3) 24 (23.3) 144 320 384 160 144 144

B20 <4 <4 <4 <4 (27.0) <4 (24.5) 4.5 (19.0) 72 320 160 160 128 160 192 96 144

Legend: The numbers represent the virus neutralising antibody titre, the boxes shaded in grey indicate the days when individual animals were viraemic, and the numerals in parentheses are the positive Ct values obtained by qRT-PCR. Virus neutralising antibody titres ≥4 are considered positive. Note: In all the four animals, viraemia was first detected on day 4.

2.4.2.2 Kinetics of plasma IL-10, IL-6, IL-2, and IFN-γ

During the nine days of cytokine screening of the second cohort of study cattle, plasma IL-10 values in individual virus-infected animals were consistently greater than those observed in the negative controls (Figure 2.3). The increase in IL-10 was already observed on day 1 of sampling which was three days prior to the detection of viraemia in all the four animals. When the overall plasma cytokine expression trends were evaluated, plasma concentrations of IL-10 for animal B05 were slightly higher than for the other three virus-infected heifers for the larger part of the nine days with only minor exceptions.

Figure 2.3: Kinetics of plasma IL-10 in four adult Brahman cattle infected with the bovine ephemeral fever virus and four uninfected negative controls. Note that the onset of viraemia indicated with a thick arrow was first detected on day 4 in all the four animals.

In respect to plasma IL-6 concentrations, the individual animal values were also generally higher in virus-infected animals than in the negative controls and appeared to pick momentum around day 3, which was a day prior to the detection of viraemia.

Figure 2.4: Kinetics of plasma IL-6 in four adult Brahman cattle infected with the bovine ephemeral fever virus and four uninfected negative controls. Note that the onset of viraemia indicated with a thick arrow was first detected on day 4 in all the four animals.

Regarding the kinetics of plasma IL-2, the OD450nm readings for animal B20 were consistently greater than for the other three virus-infected animals plus the four negative controls throughout the 9 days of cytokine studies (Figure 2.5). With the exception of the OD450nm readings for animal B10 that were slightly above the negative mean on days 4, 7, and 9, the overall IL-2 values for animal B05, B10, and

B11 were comparable with the negative control mean. Interestingly, of the four virus-

37 infected heifers, B11 that showed the lowest IL-2 and IFN-γ OD450nm readings, demonstrated the longest viraemia (Table 2.4; Figure 2.5; Figure 2.6). When the

OD450nm readings were considered as an index of IFN-γ production, animal No B20 with the shortest viraemia had IFN-γ production indices that were consistently greater than for the other three virus-infected animals (Table 2.4; Figure 2.6).

Overall, the IFN-γ production indices for this animal were consistently higher than the negative control mean throughout the nine days of cytokine screening (Figure 2.6).

In animal B10, the IFN-γ readings gradually started increasing and exceeded the negative control mean from around day 4 when viraemia was first detected through day 9 (Figure 2.6).

Figure 2.5: Kinetics of plasma IL-2 in four adult Brahman cattle infected with the bovine ephemeral fever virus and three uninfected negative controls. Note that the onset of viraemia indicated with a thick arrow was first detected on day 4 in all the four animals.

Figure 2.6: Kinetics of plasma IFN-γ in four adult Brahman cattle infected with the bovine ephemeral fever virus and three uninfected negative controls. Note that viraemia was first detected on day 4 in all the four animals.

2.5 DISCUSSION

As one of the objectives, the present study set out to evaluate the relationship between fever, seroconversion, and resolution of viraemia in BEFV-infected cattle.

On the first day of clinical monitoring, two of the three BEFV-infected heifers were clinically ill, recumbent, and pyrexic while the third animal was pyrexic on day 3.

However, the fever in in the second and third animals subsided by the second day of clinical monitoring. The transient nature of these clinical signs in the two virus- infected febrile heifers is consistent with classic BEF signs. As the mean incubation

40 period of BEF in cattle is about three to five days, it is probable that the first fever peak occurred prior to the first day of clinical monitoring.

The other focus of the study was to evaluate whether neutralising antibodies were the only important factor required for the resolution of the viraemia during acute to subacute BEF. Previously, Vanselow et al., (1985) reported that virus neutralising antibody titres ≥4.5 were protective against BEFV in cattle. According to data obtained in this study, however, it is apparent that in vivo virus neutralisation in

BEFV-infected cattle may occur over a wider antibody titre range than previously suggested. Moreover, the viraemia in two of the febrile heifers promptly resolved when the neutralising antibody titres reached 8 and 16, respectively. In the other virus-infected animal, however, viraemia continued for three days after seroconversion and only subsided when the antibody titre reached 72. While these observations underscore the important role that virus neutralising antibodies appear to play in the anti-BEFV immunity, the present data also suggest that additional, and uncharacterised immune mechanisms, may be required for the resolution of viraemia during BEFV infections in cattle.

The presumed role of IL-1β in the pathogenesis of BEF was initially suggested by

Uren and Zakrzewski (1989) based on the limited studies they conducted in BEFV- infected cattle. Prior to the studies reported by these authors (Uren and Zakrzewski,

1989), experimental injection of cattle with recombinant bovine IL-1β had shown transient BEF-like clinical signs that included pyrexia, increased pulse rate, and an elevated respiratory rate (Goff et al., 1992). Like other pyrogenic cytokines, IL-1β is known to upregulate the enzyme cyclooxygenase-2 (Cox-2), which is subsequently

41 followed by release of prostaglandin-E2 (PGE2) that ultimately induces fever (Feghali and Wright, 1997; Dinarello, 2015). While signal transduction via Toll-like receptors

(Dinarello, 2004) and C5a-mediated PGE2 upregulation (Blatteis, 2007) may cause fever that is independent of the Cox-2-PGE2 pathway, studies have previously shown transient BEF-like symptoms in experimental cattle that were injected with recombinant bovine IL-1β (Goff et al., 1992). These studies aside, Uren et al. (1989) abrogated fever in BEFV-infected cattle by treating them with the non-steroidal anti- inflammatory drug phenylbutazone. As the latter is a well-known Cox-1/2 inhibitor, it is hence implied that Cox-1/2-PGE2 pathway-dependent IL-1β signalling may be important in the pathogenesis of inflammation and fever during acute BEF. It is noteworthy that in the present study, the plasma IL-1β expression kinetics in the

BEFV-infected febrile heifers was comparatively higher than in the negative controls

(Figure 2.1A) suggesting that IL-1β, which is a well-known pyrogenic cytokine, contributed to the fever response seen in two of the virus-infected animals on day 1.

However, this does not preclude involvement of IL-1β in other signalling pathways

(Blatteis, 2007; Dinarello, 2004) since continuous upregulated expression of IL-1β and the other proinflammatory cytokines in the virus-infected heifers did not translate into fever beyond day 1.

Overall, the expression kinetics of TNF-α in virus-infected animals were comparatively lower than for the other three cytokines (Figure 2.1B). However, plasma TNF-α concentrations on days 3, 5, and 6 were higher in the BEFV-infected heifers than in the negative controls peaking on days 3 and 5, well after the day 1 fever response in two of the three virus-infected heifers. While peaking of plasma

TNF-α concentrations was not associated with a detectable pyrexic response in the

42 present study, the biphasic expression pattern was not kinetically dissimilar to the polyphasic fever that would have been expected. For unclear reasons, however, the mean plasma TNF-α concentration in the three virus-infected animals on day 2 was comparatively lower than the negative control mean. It should be noted that besides being a pyrogen, TNF-α has pro-inflammatory properties mediated through activation of antigen processing and presenting cells as well as stimulation of the up-regulation of adhesion molecules and chemokines on endothelial cells and leukocytes

(Feldmann et al., 1997; Kushibiki, 2011). As such, the relative increase of plasma

TNF-α seen in the BEFV-infected heifers suggests some role(s) of this cytokine in the pathogenesis of BEF which, speculatively, may include inflammation and immune activation. Due to the small sample size of this study that was inevitable since were based on natural BEFV infections, elaborate and larger scale studies would be needed to further evaluate this hypothesis.

Individual plasma IL-6 concentrations were also consistently higher in the three

BEFV-infected than in the negative control animals (Figure 2.1C). By day 1, the increase in IL-6 plasma levels was already underway followed by a minor decline between days 3 and 4 before rising slightly through days 5 and 6 (Figure 2.1C).

Besides the pyrogenic properties, IL-6 promotes innate and adaptive immune mechanisms (Kishimoto et al., 1992; Yang et al., 2016) that may potentially contribute to the resolution of viraemia as speculated on the basis of the data obtained in the present study. On the other hand, the kinetics of plasma IL-6 somewhat mirrored that of IL-10 with the two cytokines apparently equilibrating around the time of seroconversion (Figure 2.1; Figure 2.2D). Considering the shared properties of IL-6 and IL-10 in B-cell activation and differentiation (Aggarwal et al.,

2017; Houssiau et al., 1988; Kishimoto et al., 1992; Yang et al., 2016), and if interpreted in the context of the present study, the two cytokines most likely played some role(s) in pre-seroconversion cellular events that may have ultimately paved the way to the antibody production in the BEFV-infected cattle.

Similar to what was observed for IL-1β and IL-6, individual plasma IL-10 concentrations were generally higher in the three BEFV-infected heifers than in the negative controls and increased expression was already underway by day 1 (Figure

2.1D). As the mean incubation period of BEF is three to five days, it is possible that the viral infection in the three heifers had been present for up to or greater than five days by the time viraemia was detected. It should be expected that within that timeframe, cellular and other innate immune mechanisms like those mediated by type I interferons would have been underway in the virus-infected heifers. However, while IL-10 may enhance B-cell proliferation and differentiation (Chen and Zlotnik,

1991; Choe and Choi, 1998; Demeure et al., 1997), human-based studies have also shown that this cytokine may also potentially suppress the production of proinflammatory cytokines (Demeure et al., 1997; Fiorentino et al., 1991; Harden et al.,

2013; Murthy et al., 2000). In the first cohort of cattle, fever was observed in two of the three virus-infected heifers only on day 1 (Table 2.2) and plasma IL-10 concentrations were consistently higher in the infected than in the negative controls

(Figure 2.1D). Since these studies were required to be based on natural BEFV infections, this inevitably led to many animals not meeting the inclusion criteria. As the sample size at the end was not statistically representative, whether or not increased IL-10 production down-modulated the fever response in BEFV-infected

44 animals is not clear. It is therefore recommended that further studies are conducted on this phenomenon using a larger sample size.

The major objectives of the study based on the second cohort of cattle were to describe the kinetics of IL-2 and IFN-γ (‘Th1 indicator cytokines’) along with IL-6 and

IL-10 during the innate-immune response transition in adult cattle infected with

BEFV. In addition, this part of the study evaluated the relationship between the virus neutralising antibody response and the duration of viraemia. As the study data show, cessation of viraemia in 3/4 virus-infected animals promptly occurred upon seroconversion. The limited sample size notwithstanding, this observation underscores the role played by virus neutralising antibodies in resolving viraemia in

BEFV-infected cattle as has previously reported (Aziz-Boaron et al., 2013; Uren et al., 1994). Interestingly, viraemia in the fourth virus-infected animal continued for a total of six days, the last two days of which were in presence of virus neutralising antibody titres >4.5, deemed protective for cattle (Vanselow et al., 1985). Moreover, several studies have also shown that some cattle inoculated with inactivated BEFV vaccines may succumb to challenge with BEFV in the presence of high anti-BEFV neutralising antibody titres (Della-Porta and Snowdon, 1979; Aziz-Boaron et al.,

2014; Tzipori and Spradbrow, 1973). While virus neutralising antibodies are certainly important in resolving viraemia in BEF (Aziz-Boaron et al., 2013; Uren et al., 1994), data hereby presented suggest that additional mechanisms may also be required.

To further elucidate on the cytokine events that prevail during seroconversion and progression of the virus neutralising antibody response in BEF, the kinetics of IL-6 and IL-10 were also evaluated in the second cohort of cattle. On the basis of the data obtained, upregulated IL-10 production in virus-infected animals was already

45 detectable on day 1 which was three days prior to the actual detection of viraemia in all the four virus-infected animals (Barigye et al., 2015). As the mean incubation period in BEF is reported to be three to five days or longer (St. George, 1986), it is evident - on the basis of these and data from the first cohort of cattle - that increased

IL-10 production occurs earlier on during BEFV infection, apparently preceding seroconversion by several days. Since IL-10 is known to induce B-cell activation and differentiation (Choe and Choi, 1998; Wang et al., 2016; Zhang et al., 2015), interpreted in the context of the data as obtained from both the first and second cohorts of study cattle, IL-10 cytokine is indeed likely to play a role(s) in pre- seroconversion immunological events that may prevail during BEFV infections in cattle. On the other hand, increased plasma IL-6 somewhat lagged behind the IL-10 response in BEFV-infected animals and only started increasing around day 3, a day before the detection of viraemia, followed by a visible peak around day 6. Within the limits the present data may permit, the precise role(s) played by IL-6 in the pathogenesis of BEF is at best inconclusive. However, it is known that this cytokine has multiple, pleiotropic effects that are proinflammatory, pyrogenic, as well as immunoregulatory (Coussens et al., 2004; Feghali and Wright, 1997; Houssiau et al.,

1988; Kishimoto et al., 1992; Yang et al., 2016) so its role in the pathogenesis of

BEF in cattle requires additional studies. Nevertheless, while fever has not been recorded in any of the study animals from the second cohort, both inflammation and pyrexia tend to be common features in BEFV infections in cattle (Uren et al., 1989).

Besides, the overall consistency in the upregulation of plasma IL-6 and IL-10 during the pre-seroconversion period in the virus-infected animals was similar in both cohorts of studied cattle. The present data underscore the relevance of both IL-6 and

IL-10 in the mechanisms that initiate and perpetuate the humoral adaptive immune

46 response in BEF as suggested by the kinetics and sustained antibody response during the post-seroconversion period.

In respect to the kinetics of ‘Th1 indicator cytokines’, animal B20 showed comparatively higher IL-2 and IFN-γ production indices than the other three virus- infected heifers plus the four negative controls. Interestingly, this animal (B20) also demonstrated the shortest viraemia of three days. On the contrary, animal B11, which showed the lowest plasma IL-2 and IFN-γ production indices demonstrated the longest viraemia, despite adequate virus neutralising antibody titres of 4.5 and 24 during the last two days of BEFV detection, respectively. Despite the small sample size, these findings suggest that resolution of viraemia during BEFV infections may require both virus neutralising antibodies (Aziz-Boaron et al., 2013; Barigye et al.,

2015) and additional mechanisms that presumptively may be mediated or/and associated, at least in part, to IL-2 and IFN-γ. Other than the innate antiviral systems like those associated with type I interferons (IFN-α/IFN-β), non-humoral based immunological mechanisms may involve activation of NK cells and macrophages as well as adaptive immune mechanisms like those related to activation of CD8+ T-cells

(Ehl et al., 1997; Goodbourn et al., 2000; Weinstein et al., 2017; Whitmire et al.,

2005). Since high plasma IL-2 and IFN-γ indices were accompanied by rapid resolution of viraemia in one animal, it is likely that CD4+ and CD8+ T-lymphocyte responses and related cellular mechanisms may stimulate the virus neutralising antibodies in resolving the viraemia in BEF. However, the IFN-γ in this study could have been part of the innate immune response since NK and other cells may also produce this cytokine (Feghali and Wright, 1997; Weinstein et al., 2017). It is noteworthy that in vitro proliferation of antigen specific T-cells derived from cattle with prior immunisation with a BEFV-derived immunogen was associated with

47 increased IL-2 production (Uren et al., 1993). Interleukin-2 is an important growth factor for T-cells, NK cells, and B- cells and promotes the activation of lymphokine- activated killer cells (Arai et al., 1992; Jounaidi et al., 2011; Romanova et al., 2017).

Against this background, the remarkably high IL-2 production indices in the heifer that manifested the shortest viraemia suggests that this IL-2 may possibly contribute to innate and adaptive immune responses required to resolve BEFV infections.

Besides this, IFN-γ produced by a variety of cells including NK cells and activated

CD4+ helper lymphocytes stimulates immunoglobulin isotype switching (Estes et al.,

1994; Weinstein et al., 2017) while also promoting activation of and enhanced killing by macrophages (Adler et al., 1994; Stich et al., 1998). In addition, IFN-γ may cause activation of NK and cytotoxic T-cells, which are also important in the antiviral immunity (Arai et al., 1992; Weinstein et al., 2017). Against this background, it may be inferred that IFN-γ-mediated mechanisms are important for the successful resolution of viraemia during BEFV infections in cattle. It is also noteworthy that 2/3 of the virus-infected heifers had baseline IL-2 and IFN-γ production indices comparable to the negative controls. Interestingly, these were the animals with the longest viraemia.

2.6 CONCLUSIONS

Based on the data from the first cohort of cattle, IL-1β was more prominently expressed in BEFV-infected cattle during the febrile phase of the infection than IL-6,

IL-10 or TNF-α in a descending order. Evaluation of these data suggested that IL-1β is comparatively more associated with pyrexia in the BEFV-infected cattle. Further still, data from the second cohort of cattle demonstrated that increased production of

IL-10 and IL-6 precedes seroconversion during BEFV infections in cattle. Interpreted

48 together, data on the kinetics of plasma IL-6 and IL-10 during the pre-seroconversion period suggest the two cytokines could have potentially contributed to humoral immunity which may have been mediated by mechanisms that could have involved

B-cell activation and differentiation. On the basis of the overall data pertaining to the kinetics of IL-10, the potential modulatory role of IL-10 on the fever response in

BEFV-infected cattle is suspected but needs to be further investigated. While viraemia in the first cohort of study cattle promptly resolved within one day of seroconversion in two animals, the persistence of viraemia three days after seroconversion in the other virus-infected heifer suggests additional but yet undefined antiviral mechanisms in BEF. Comprehensive cellular immunology studies are needed to further elucidate on the contributory role of T-cell immunity in anti-

BEFV immunity in cattle. Knowledge from such studies would inform alternative vaccine design strategies by providing a rationale for incorporating T-cell epitopes in vaccine candidate antigens. While remarkable variability was observed in the expression of plasma IL-2 and IFN-γ in the second cohort of virus-infected cattle, the present research findings suggest that increased production of IL-2 and IFN-γ may shorten the duration of viraemia in BEFV-infected cattle. Presumptively, the two Th1 indicator cytokines may arguably achieve this through activation of effector cellular mechanisms that augment virus neutralising antibodies in removing circulating

BEFV. Despite the small sample size which was an inevitable weakness of the study

(of the total of 42 cattle from both cohorts 1 and 2, only 14 met the inclusion criteria), the present findings are likely to reflect the true protective immune response since these studies were based on natural field BEFV infections. To further evaluate the validity of the hypotheses generated in this research, the author of this thesis recommends specific studies to further define the kinetics of CD4+ and CD8+ T cell

49 responses alongside a broader range of Th1 and Th2 cytokines in a larger group of

BEFV-infected cattle.

CHAPTER 3

TISSUE TROPISM AND POTENTIAL REPLICATION SITES OF BOVINE

EPHEMERAL FEVER VIRUS IN CATTLE

This chapter is a composite of data published as a full research article, case report, and a conference paper. For consistency of formatting within the thesis and to minimise self-plagiarism concerns, modifications have been made to the already published text.

1. Research paper published in the Australian Veterinary Journal Barigye, R1,2,3, Davis, S.1, Hunt, R. 1, Hunt, N. 1, Walsh, S.1, Elliott, N.1, Burnup, C.1,

Aumann, S.1, Day, C.1, Dyrting, K.1, Weir, R.1, Melville, L.F1. Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle, Australian Veterinary Journal, 2016, Vol 94, pp.

362-370.

2. Case report published in the Australian Veterinary Journal

Barigye, R1,2,3, Davis, S1, Hunt R.1, Hunt, N.1, Walsh, S.1, Elliott, N.1, Dyrting, K.1,

Weir, R.1, Melville, L.F.1 Post-viraemic detection of bovine ephemeral fever virus by use of autogenous lymphoid tissue-derived bovine primary cell cultures in the

Australian Veterinary Journal, 2017, Vol 49, pp. 49-52.

3. Conference abstract published in the Journal of Clinical and Experimental

Pathology:

Barigye, R1,2,3, Burnup, C1, Davis, S1, Aumann, S1, Hunt. R1, Hunt, N1., Walsh, S1.,

Melville L.F1. Application of immunohistochemistry and quantitative real time

51 polymerase chain reaction to the study of tissue virus tropism in adult cattle infected with the bovine ephemeral fever virus, Journal of Clinical and Experimental

Pathology 2016, 6:3

1Berrimah Veterinary Laboratories, Department of Primary Industry and Resources,

GPO Box 3000, Darwin, NT 0801, Australia

2School of Veterinary Science, University of Queensland, Gatton QLD 4343,

Australia

3Department of Veterinary Medicine, College of Food and Agriculture, United Arab

Emirates University, PO Box 15551, Al Ain, UAE

3.1 ABSTRACT

While several studies have described clinical and pathological aspects of BEF, major gaps still remain in our understanding of the pathogenesis of the disease in cattle. In particular, the potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of a BEFV infection have not been defined in cattle. To clarify these knowledge gaps, the present studies evaluated the overall tissue tropism and potential replication sites of BEFV in adult cattle. Various specimens from nine adult cattle that died or were euthanased at different time points following natural BEFV infections were tested for viral antigens and RNA by

IHC and qRT-PCR, respectively. Of the samples taken from the nine animals, the spleen and haemal node from the steer necropsied 6 days after acute BEF were additionally tested by a transmission electron microscope (TEM). Overall, virus antigen was detected in several tissues/organs in all the study cattle and both virus antigen and RNA were simultaneously demonstrated in the spleen and/or haemal

52 node from 7/9 cattle. The longest period of RNA detection was in the haemal node

120 days following the initial qRT-PCR-based BEF diagnosis. In all tissues where the viral antigen was detected, which included but were not limited to spleen, haemal node, lymph node, viral proteins were intracytoplasmic within cells morphologically consistent with macrophages, neutrophils, dendritic cell-like cells and spindle shaped cells of perivascular location. Significant necropsy findings in the case study animal included fibrinoproliferative synovitis in the stifle joints and fibrin clot-laden fluid in serous body cavities. Moderate numbers of infiltrating neutrophils were demonstrated in sections of the prefemoral lymph nodes and haemal node, and lymphoid hyperplasia in the spleen, haemal node and prefemoral lymph nodes. In the case study animal euthanased 6 days after cessation of the viraemia, viral RNA was detected by qRT-PCR in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in several spleen-derived cell cultures. Furthermore, BEFV was isolated from autogenously derived splenic primary cell cultures established from 6 days after cessation of viraemia, and characteristic bullet-shaped virions confirmed by electron microscopy in ultrathin sections from the haemal node. Through demonstration of virus antigens in various tissues, this study has elucidated on the overall tissue tropism of the BEFV in cattle. Based on these data, BEFV appears to have preferential tropism for bovine lymphoid tissues. In particular, spleen and haemal node may be potential sites for viraemic and post-viraemic virus replication.

3.2 INTRODUCTION

While several studies have described the clinical and pathological aspects of BEF,

(Basson et al., 1970; Burgess and Spradbrow, 1977; Mackerras et al., 1940; Young and Spradbrow, 1990), major gaps still remain in our understanding of the

53 pathogenesis of the disease in cattle. Previously, the fluorescent antibody test was used to demonstrate intracellular viral antigens in tissues and within cellular components of serosal fluids taken from BEFV-infected cattle (Burgess and

Spradbrow, 1977; Young and Spradbrow, 1985; Theodoritis, 1969). In one of these studies (Burgess and Spradbrow, 1977), viral antigens appeared to be located intracytoplasmically within neutrophils as well as reticular cells of spleen and lymph nodes. Despite these findings, however, the tissue tropism and potential in vivo replication sites of the virus remain undefined to date.

Immunohistochemistry (IHC) is a powerful tool that has been extensively used in disease pathogenesis studies as well as in the diagnosis of infectious diseases

(Baszler et al., 1995; Nakamura et al., 2008). When used alongside other molecular techniques like PCR, IHC is a potentially powerful tool that is suitable for determining the spatial distribution of microbial antigens in tissue samples (Barigye et al. 2016).

For these reasons, both IHC and real time PCR were chosen as the techniques of choice to address the study objectives of this part of the thesis, as follows: (a). to determine the tissue tropism of BEFV, and (b) to elucidate on the potential tissue replication sites of the virus in cattle. The study used biological specimens derived from adult cattle naturally infected with BEFV along with uninfected BSR cell lines plus BSR and MDBK cell lines infected with BEFV and six other viruses namely

BRMV (Gard et al., 1983), KIMV (Cybinski and Zakrzewski, 1983), ADRV (Gard et al., 1984), BoHV-1 (Snowdon, 1964), AKAV (Doherty et al., 1972), and BTV-1 (St

George et al., 1980).

3.3 MATERIALS AND METHODS

3.3.1 Experimental cattle

Blood samples were taken for obtaining serum and plasma from a total of nine adult

Brahman cattle at different time points following the initial qRT-PCR-based confirmatory BEF diagnosis. Following euthanasia, BEFV-infected tissues, and other biological specimens like synovial fluids, pericardial fluids, peritoneal fluids, were also taken at the time of necropsy examination. All the nine animals were naturally infected with BEFV and belonged to sentinel herds that were being used for routine

Arbovirus monitoring and surveillance at different research farms owned by the

DPIR, NT Government. Of the nine animals, one died from the acute BEF a day after a confirmatory BEF diagnosis while the other eight were humanely euthanised on days 1, 7, 23, 30, 60, 93, 120, and 150 after the initial BEF diagnosis (Animal Ethics

Committee approval notice No A12028). The animals euthanised on days 23 (No 4) and 93 (No 7) were downer cattle with paralysis as a complication of clinical BEF while the rest of the animals were euthanased as part of a systematic study protocol that sought to evaluate post-viraemic localisation of BEFV in cattle tissues. The four neurologically affected animals were ethically treated and cared for as stipulated in theAnimal Ethics Committee approval notice no. A12036. This included nursing them under a shade, providing feed and water, and frequently gently assisting them in their attempts to stand. The severely paralysed animals No 4 (euthanased at day 23) and No 6 (euthanased at day 42) were propped up with bales of hay. When recovery was considered unlikely, the animals were euthanised with intravenous barbiturates.

On the days of necropsy, samples of jugular blood were taken from the live animals prior to induction of euthanasia by intravenous infusion of Lethabarb (Animal Ethics

Committee approval notice No A12028). Various biological specimens including

55 body fluids and fresh and formalin-fixed tissues were collected from all the nine animals during post mortem examination. The body fluid samples included synovial, pericardial, thoracic, and peritoneal fluids, while tissue samples included liver, lung, kidney, urinary bladder, spleen, haemal node from subcutaneous locations of the thoracoabdominal area, heart, skeletal muscle from the hamstring muscle group, tongue, synovial membrane (stifle joint and carpal joints), prefemoral lymph node, peripheral nerve (gluteal nerve in all animals and gluteal and fibular nerves plus nerves taken from the left and right branchial plexus in the case study animal), spinal cord (cervical, thoracic, and lumbar segments), cerebrum, cerebellum, and medulla oblongata. As negative controls for IHC, qRT-PCR-negative specimens from an adult Brahman cow that died from shipping fever were used. These included freshly collected formalin-fixed spleen, heart, lung, and kidney. In addition, blood and post- mortem tissue samples from an adult male sheep were collected and also used as additional negative controls.

3.3.2 Virus neutralisation test and virus isolation

The virus neutralisation test was done on heat-denatured serum samples (n=9) using a modified version of the BEFV neutralisation test protocol initially described by Uren et al. (1989). The test was done in 96 well plates as recently reported by Barigye et al. (2015) and the neutralising antibody titres calculated using the 50% end point method (Reed and Muench, 1938). To isolate BEFV from the blood samples, each jugular EDTA blood specimen was centrifuged at 4000 RPM for 10 min and the buffy coats collected and inoculated onto monolayers of C6/36 Aedes albopictus

(mosquito/insect) cells held in tubes containing 2 mL Minimum Essential Medium

(MEM) containing Earle’s salts and 10% foetal calf serum (FCS). After 24 h of

56 incubation at room temperature, the medium was removed and replaced with fresh

MEM. After 14 days of incubation at room temperature, the cell cultures were sonicated for 30 min, the supernatants collected and inoculated onto BSR monolayers (BSR is a subclone of the hamster cell line BHK-21), and finally incubated at 37◦C in cell-culture flasks containing Eagle’s basal medium, pH 7.2, that was supplemented with 10 mM HEPES, 6.7 mM NaHCO3, 2mM L-glutamine, 80 U penicillin, 137 mM streptomycin and 5% FCS. Thereafter, the cell cultures were examined daily for cytopathic effects (CPEs) for 7 days. In cases where CPEs were not observed during the first 7 days, supernatants of the cultures were re-inoculated onto fresh BSR monolayers and examined for additional 7 days.

3.3.3 qRT-PCR for BEFV

The qRT-PCR protocol used in the present study was previously standardised for the detection of a fragment of the conserved region of the glycoprotein (G) gene located within the BEFV genome. Viral RNA was extracted from study samples (tissue samples, blood, pericardial, thoracic and peritoneal fluids) using the MagMAX-96 viral RNA isolation kit (Applied Biosystems, CA, USA) according to the manufacturer’s instructions and the qRT-PCR assay performed as described under

2.3.2 above. The primer sequences and concentrations used in the assay are presented in Table 2.1 above and detailed information on negative and positive controls are provided under appendix 3.

3.3.4 Tissue processing and histology

Fresh tissue specimens were fixed in 10% buffered formalin, routinely processed and paraffin embedded. Four-micron sections were deparaffinised, stained with

57 haematoxylin and eosin and evaluated for microscopical lesions at low ad high magnification using a light microscope (Olympus-BX41, Model U-MDOB3).

3.3.5 Autogenously-derived splenic and haemal node cell cultures

Specimens of lymphoid tissue (spleen and haemal node) collected from the paralysed case study steer euthanised at 6 days following the initial BEF diagnosis were placed into sterile Gibco® Medium 199 (Life Technologies, NY, USA), autogenous cell cultures established, and used for virus isolation. The aseptically collected spleen and haemal node were separately processed according to an in- house protocol and the autogenous primary bovine cell cultures derived from the two organs were established and periodically tested by qRT-PCR to assess the status of

BEFV infection. To prepare the cell cultures, the spleen and haemal node were separately macerated and mechanically agitated to yield fine, cell-rich slurries which were then mixed with a small amount of 0.12% trypsin (SAFC, Sigma-Aldrich, St

Louis, MO, USA) and homogenised by additional mechanical agitation. The resulting cell-rich homogenate was mixed with Gibco Medium 199 supplemented with bovine pituitary-derived fibroblast growth factor (Sigma-Aldrich) to a final concentration of

3.2 ng/mL. Next, 10 mL of the cell suspension were dispensed into 25-cm2 tissue culture flasks, yielding a total of 16 spleen and 4 haemal node derived level 0 passages (P0) that were incubated at 37ᵒC. After 24 h of incubation, the flasks were washed with unsupplemented Medium 199, replenished with fresh medium supplemented with bovine pituitary-derived fibroblast growth factor at 10 ng/mL

(Complete Medium 199) and then further incubated at 37ᵒC. At days 8, 15 and 21 of incubation, small aliquots of P0 tissue culture medium were tested for BEFV by qRT-

PCR. On day 15, 4/16 splenic cultures with confluent cell monolayers were

58 passaged and level 1 (P1) cultures established. Briefly, the culture medium was removed and the cells washed with 10 mL of phosphate-buffered saline after which they were treated with 1 mL of 0.12% trypsin. The monolayers were then removed and resuspended in Complete Medium 199 held in 25-cm2 flasks or chamber slides that were then incubated at 37ᵒC. After 24 h, the medium was removed from one flask, an aliquot of the supernatant tested for BEFV by qRT-PCR, the cells pelleted by centrifugation (and the medium on the chamber slides decanted), fixed for 1 h in

10% buffered formalin and then processed for BEFV-IHC. For immunocytochemical evaluation of the autogenously derived splenic and haemal node cell cultures, processed chamber slides and 4-μm sections of paraffin-embedded cell pellets were also tested using the same protocol. After 21 days of incubation, aliquots of the spleen and haemal node tissue culture supernatants from all cultures were again tested by qRT-PCR (Table 4.2).

3.3.6 Paraffin-embedded blocks of virus-infected cell cultures

Paraffin-embedded blocks of uninfected BSR cells plus cell lines separately infected with BEFV and six other viruses were prepared and used as controls for IHC. The parent stocks of the seven viruses are currently held in cryopreservation at the BVL,

Darwin, NT, Australia and include the W138 strain of the BEFV originally received from the Veterinary Research Station, Glenfield, QLD Australia (Littlejohns I, 1979, unpublished data); and three vector-borne rhabdoviruses closely related to the BEFV including the DPP63 strain of BRMV (Gard et al., 1983) the CSIRO368 strain of

KIMV (Cybinski and Zakrzewski, 1983), and the DPP61 strain of ADRV (Gard et al.,

1984). In addition, paraffin-embedded blocks of cell cultures infected with the V155 strain of BoHV-1 (Snowdon, 1964), the R7949 strain of AKAV (Doherty et al., 1972),

59 and the CSIRO156 strain of BTV-1 (St George et al., 1980) were also used to specifically rule out cross-reaction between BEFV and these viruses. With the exception of BoHV-1, which was inoculated into MDBK cells, all the other viruses including BEFV, AKAV, BTV-1, BRMV, ADRV and KIMV were grown in BSR cell lines.

3.3.7 Immunohistochemistry

Immunohistochemistry (IHC) was performed using a Dako EnVision + System-HRP

(AEC) kit (Dako Australia Pty Ltd, Campbellfield VIC, Australia) according to a protocol initially standardised and reported by Barigye et al. (2013) and Barigye et al.

(2016). Tissues tested by IHC included but were not limited to spleen, haemal nodes, skeletal muscle, cardiac muscle, liver, lung, kidney, synovial membrane, spinal cord, brain, and peripheral nerves. The spleen from a Droughtmaster bull positive for BEFV by qRT-PCR was used as apositive control while the negative controls were splenic tissues from a BEFV-negative heifer, a male sheep, as well as paraffin-embedded uninfected BSR cells trimmed at 4µm. Duplicate sections of test tissues per block as well as the positive and negative controls were adhered onto salinized slides (FLEX IHC microscope slides, Dako),deparaffinised in two steps of histolene, re-hydrated through three grades of absolute ethanol, and equilibrated in

Tris buffered saline-Tween 20, pH 7.4 (TBS/T pH 7.4) for 5 min. The primary mouse monoclonal antibodyDB5 (Cybinski et al., 1992) and the negative control mouse serum were diluted at 1:1500. The mAb is specific for epitopes located within the G1 site of the viral envelope glycoprotein of the BEFV and has been previously used to characterise bovine-derived ephemeroviruses (Cybinski et al., 1992; Walker et al.,

1991). Briefly, after equilibrating in TBS/T pH 7.4, the sections were incubated with

0.03% hydrogen peroxide (Peroxidase block; Bottle 1) for 15 min at room temperature (RT). After rinsing in TBS/T pH 7.4, the sections were separately incubated for 30 min at RT with the mAb and negative mouse serum diluted at

1:1500 in DAKO antibody diluent solution (Cat. No S0809). The slides were then washed with TBS/T pH 7.4 and further incubated for 30 min in RT-equilibrated horse radish peroxidase-labelled polymer conjugated to goat anti-mouse IgG (Bottle 2 of the DAKO kit). After washing the slides with TBS/T pH 7.4, they were further incubated at RT for another 10 min with AEC. To stop the enzymatic reaction, the slides were washed with distilled water, counterstained with Mayer’s aqueous haematoxylin for 5 min at RT, rinsed with distilled water, and then differentiated by

10 dips in Scott’s tap water. After rinsing in distilled water for a further 5 min, the slides were air dried and mounted with Glycergel. The immunohistochemical evaluation was performed under a light microscope (Olympus-BX41, Model U-

MDOB3) and photomicrographs taken by means of an imaging software Olympus

Labsens.

3.3.8 Transmission electron microscopy

To examine the presence of BEFV in the tissues and its morphology, ultrathin sections of spleen, and haemal node were examined by transmission electron microscopy. Briefly, tissue blocks originally fixed in 10% formalin were recovered, trimmed into ~5 mm x 2mm blocks which were blot dried and then fixed in 2.5% (v/v) glutaraldehyde in 0.1M cacodylate buffer (pH 7.4, 300 milliosmoles). This was followed by washing the blocks with 0.1 M cacodylate buffer after which they were fixed with 1% (w/v) osmium tetroxide. After three 5 min washes in distilled water, the specimens were dehydrated in increasing concentrations of ethanol starting from

30% through to 100%. After dehydration, the specimens were infiltrated with graded series of ethanol: Spurr’s resin (30%, 50%, 70%, 100%). The tissue specimens were then embedded in 100% Spurr’s resin, which was allowed to polymerise overnight at

60°C. Tissue blocks were then trimmed, and ultra-thin sections cut using a Leica

(Reichert-Jung) Ultracut 2E. The ultrathin sections of spleen and haemal node were then mounted on Parlodion-filmed and carbon-coated grids and stained with uranyl acetate followed by lead citrate and then examined at 80 kV using a JEOL 1200SX transmission electron microscope at the Charles Darwin University.

3.4 RESULTS

3.4.1 Virus neutralisation test and virus isolation

At the time of the initial qRT-PCR-based diagnosis, eight out of nine virus-infected animals were seronegative. However, by the time of post-mortem examination, the virus neutralising antibody titres for eight animals were positive (>4.5) with the exception of the ninth animal (Table 3.1). This was despite the fact that the seronegative animal had seroconverted shortly after the initial BEF diagnosis. In addition to the serological findings, BEFV was isolated from the peripheral blood of two animals on the day of the initial BEF diagnosis, and from peripheral blood, spleen, haemal node, and lymph node collected during post-mortem examination of the heifer that died from acute BEF. Interestingly, BEFV was not isolated from peripheral blood, spleen, haemal node, and skeletal muscle of the heifer that was euthanised three weeks after the initial BEF diagnosis despite these specimens being qRT-PCR-positive for viral RNA on the day of post-mortem examination.

Regarding the autogenously derived splenic and haemal node cultures from the case study steer, adherent multiplying cell colonies were found in all cultures after

62 incubation of each passage level, with the majority of cells having a typical spindle- shaped morphology consistent with fibroblasts (Fig. 3.1). Interestingly, BEFV was successfully isolated in the autogenous cell lines derived from the spleen of the non- viraemic steer that was euthanised one week following the initial qRT-PCR-based

BEF diagnosis.

Figure 3.1: Splenic-derived cell cultures from the steer euthanased 7 days following a qRT-PCR-based BEFV infection diagnosis. Note that majority of the cells have a typical spindle-shape morphology consistent with fibroblasts, x40.

3.4.2 qRT-PCR results

With the exception of the prefemoral lymph node, all the tissues and body fluids from the heifer that died from acute BEF were positive for BEFV by qRT-PCR (Table 3.1).

Similarly, all the tissue and body fluid samples taken from the animal euthanised

63 during an ongoing acute BEFV infection tested positive for viral RNA (Table 3.1). In the case of the animal euthanised seven days after the initial BEF diagnosis, only the spleen, haemal node, lymph node, and synovial fluids from the stifle joints tested positive for viral RNA (Table 3.1). Interestingly, only synovial fluids from the stifle and not the carpal joints of this animal tested positive for BEFV by qRT-PCR. In the animal that was euthanised 23 days after the initial BEF diagnosis, only the spleen, haemal node, and skeletal muscle were positive for BEFV RNA (Table 3.1). In case of the three heifers euthanised 60, 93 and 120 days, the spleen, the spleen and haemal node, and only the haemal node tested positive for the BEFV, respectively

(Table 3.1). Interestingly, all tissues and body fluids taken from the animals euthanised at 30 and 150 days tested negative for viral RNA (Table 3.1). Regarding the negative controls used for IHC, peripheral blood and fresh spleen taken from the adult male sheep all tested qRT-PCR-negative for BEFV. In addition, the BEFV- infected and uninfected BSR cell lines used as the initial assay controls tested positive and negative for BEFV, respectively.

Table 3.1. Results of qRT-PCR, virus neutralisation test, and IHC tests on biological specimens collected from the nine adult

Brahman cattle with BEFV infection.

Days PCR PCR Virus Animal elapsed test on result on neutralising Immunohistochemistry Positive PCR result since jugular jugular antibody (bovine ephemeral (tissue and body fluid samples) initial blood blood (at titre at time fever virus antigen) BEF dx (acute necropsy) of BEF) necropsy (titre) All tissues (Spleen, haemal node, skeletal muscle, heart, tongue, liver, lung, kidney, urinary 1 1 + + 48 + bladder, synovial membrane, peripheral nerve, spinal cord, brain) All body fluids (pericardial, thoracic, and peritoneal fluids)

All tissues (Spleen, haemal node, skeletal 2 1 + + <4.5 + muscle, heart, tongue, liver, lung, kidney, urinary bladder, synovial membrane, peripheral nerve, spinal cord, brain) All body fluids (pericardial, thoracic, and peritoneal fluids)

Spleen, haemal node, lymph node 3 7 + - 160 + Pericardial, thoracic, and peritoneal fluids Synovial fluids (left and right stifle joints)

4 23 + - 144 + Spleen, haemal node, skeletal muscle

5 30 + - <4.5 + None

6 60 + - 48 + Spleen, haemal node

7 93 + - 72 + Spleen (haemal node not tested)

8 120 + - 32 + Haemal node

9 150 + - + (40) None

3.4.3 Histopathology

Moderate numbers of infiltrating neutrophils were demonstrated in histological sections of the prefemoral lymph node and haemal node, and moderate to marked lymphoid hyperplasia was present in sections of the spleen, haemal node and prefemoral lymph nodes of animal No. 3 (Table 3.1). In this animal, there was also a moderate fibrinosuppurative and proliferative synovitis characterised by abundant amounts of fibrin admixed with low numbers of infiltrating neutrophils occasional lymphoplasmacytic cells distributed within the affected areas of synovial tissue taken from the stifle and carpal joints.

3.4.4. Detection of BEFV in autogenous splenic and haemal node cell cultures

Viral RNA was detected by qRT-PCR in several primary cell cultures derived from the spleen but not those taken from the case study steer euthanased euthanased 7 days following the initial BEF diagnosis (Table 3.2). Note that BEFV RNA was also detected in fresh spleen, haemal node, prefemoral lymph node, synovial fluid collected during necropsy examination of this particular animal.

Table 3.2: Fractions of the different passage levels of spleen and haemal node- derived cell cultures that were positive for bovine ephemeral fever virus (BEFV) RNA by qRT-PCR.

Passage level (P) culture/ Days in culture Number of bovine primary cell cultures that

tested positive for BEFV by qRT-PCR

Spleen Haemal node

P0/day 8 8/16 0/4

P0/day 15 2/16 0/4

P0/day 21 2/12 0/4

P1/day 5 0/4 NT

P1/day 6 0/1 0/4

P1/day 7 1/11 NT

Legend: Ct value ≤45.0 = positive; NT, not tested; qRT-PCR, quantitative reverse transcription-PCR.

3.4.5 IHC detection of BEFV proteins in BSR and MDBK cell lines

Abundant intracytoplasmic viral antigen was present within many BEFV-infected cells tested with the mAB-DB5 (Figure 3.2A) while uninfected BSR cells reacted with mAB-DB5 were negative (Figure 3.22B). As would have been expected, BEFV- infected cell lines probed with negative mouse serum (Figure 3.2C) and the uninfected BSR cells reacted with the negative mouse serum (Figure 3.2D) did not demonstrate any viral antigen. In addition, KIMV-infected BSR cells separately reacted with the mAB-DB5 (Figure 3.3A), and negative mouse serum (Figure 3.3B) were both negative. All the other sections of virus-infected BSR and MDBK cell lines that were probed with either the mAb-DB5 or negative mouse serum did not show any immunostaining (Figure 3.3, Figure 3.4, and Figure 3.5). This includes sections of BSR cells infected with BRMV (Figure 3.3 C and D), ADRV (Figure 3.4 A and B),

BTV-1 (Figure 3.4 C and D), AKAV (Figure 3.5 A and B), and MDBK cells infected with BoHV-1 (Figure 3.5 C and D).

Figure 3.2: IHC for the detection of BEFV antigen done on sections of paraffin- embedded virus-infected (A and C and uninfected (B and D) BSR cells. Sections A and B were probed with the mAb-DB5 (1:1500) which detects epitopes located within the G1 site of the viral envelope glycoprotein of the BEFV while sections C and D were probed with negative mouse serum (1:1500). Note the numerous cells that demonstrate intense immunostaining of viral antigen in section A and absence of immunoreactivity in the other sections, x40 (A and B); x100 (C and D).

Figure 3.3: An IHC test for the detection of BEFV antigens was done on sections of paraffin-embedded BSR cells infected with KIMV (A and B) and BRMV (C and D).

Sections A and C were probed with the monoclonal antibody DB5 (1:1500) which detects epitopes located within the G1 site of the viral envelope glycoprotein of the

BEFV while sections B and D were probed with negative mouse serum (1:1500).

With the exception of a few cells that showed minor background staining in the

KIMV-infected cells probed with the mAb DB5 (A), all the other sections did not stain for viral antigen, x100.

Figure 3.4: An IHC test for the detection of BEFV antigens was done on sections of paraffin-embedded BSR cells infected with ADRV (A and B) and BTV-1 (C and D).

Sections A and C were probed with the mAb-DB5 (1:1500) which detects epitopes located within the G1 site of the viral envelope glycoprotein of the BEFV while sections B and D were probed with negative mouse serum (1:1500). Note absence of immunostaining in all sections, x100.

Figure 3.5: An IHC test for the detection of BEFV antigens was done on sections of paraffin-embedded BSR cells infected with AKAV (A and B) and BoHV-1 (C and D).

Sections A and B were probed with the mAb-DB5 (1:1500) which detects epitopes located within the G1 site of the viral envelope glycoprotein of the BEFV while sections B and D were probed with negative mouse serum. Note absence of immunostaining in all sections, x100.

3.4.6 IHC test for BEFV proteins in bovine tissues

Immunostaining for BEFV antigens were consistently demonstrated in sections of the spleen (Figure 3.6 and 3.7), haemal node (Figure 3.8), lung (Figure 3.9), heart (data not shown), kidney (data not shown), synovial membrane (Figure 3.10), liver (Figure

3.11) and lymph node (Figure 3.12) taken from all the eight BEFV-infected animals

73 but not in the negative control sections from uninfected animals probed with the mAb-DB5 or in the sections incubated with the mouse serum. Regarding the spleen

(Figure 3.6 and 3.7) and haemal node (Figure 3.8) from BEFV-infected cattle, viral antigens were mainly observed within the lymphoid follicles and periarteriolar lymphoid sheaths, as well as in the submesothelial areas of the capsule and the perivascular sites. The viral antigen-laden cells were morphologically consistent with macrophages, dendritic cells and pericytes. Attempts at immunophenotyping cells was then performed after the thesis review milestone, but did not yield any promising results most likely due to the antigen denaturation since the formalin tissue blocks had been stored for a long time and the lack of cross-reactivity in bovine tissue samples (positive controls in other species gave consistent results). In comparison with the other animals, the spleen and haemal node from the animal euthanased at

23 days showed comparatively more cells with intracytoplasmic antigen, as well as discrete foci of extracellular antigen scattered within the capsule, perivascular sites, and lymphoid follicles. In addition, antigen-laden cells were mainly distributed within the peripheral zones of the lymphoid follicles of the spleen and haemal node with only occasional antigen-containing cells that were present deep within the germinal centre itself. Noticeably, the lymphoid follicles of the prefemoral lymph node showed comparatively less numbers of positive intrafollicular histiocytes when compared to the spleen and haemal node. In the case of the lymph node, most of the viral antigen was restricted to spindle-shaped cells located within perivascular sites, capsule, and the connective tissue trabeculae of the medulla (Figure 3.12).

Figure 3.6: An immunohistochemically-stained section of the spleen taken from a

BEFV-affected steer euthanased 23 days after cessation of viraemia. Note the intracytoplasmic viral antigen within round cells morphologically consistent with macrophages. Note that fresh spleen, haemal node, and skeletal muscle of this animal obtained during necropsy examination also tested positive for BEFV by the qRT-PCR assay, x40.

Figure 3.7: An immunohistochemically stained section of the spleen taken from a

BEFV-affected steer 93 days after cessation of viraemia. Scattered within submesothelial areas of the splenic capsule are few macrophages that contain abundant amounts of intracytoplasmic viral antigen. In addition, very few spindle shaped cells located within the perivascular sites also contain abundant amounts of intracytoplasmic viral antigen. Note that the fresh spleen of this animal obtained during necropsy examination also tested positive for BEFV by the qRT-PCR assay, x40.

Figure 3.8: An immunohistochemically stained section of the haemal node taken from a BEFV-affected steer 1 week after cessation of viraemia. Note the intracytoplasmic viral antigen within round cells presumed to be macrophages. The fresh spleen, haemal node, lymph node, and synovial fluid of this animal obtained during necropsy examination tested positive for BEFV by qRT-PCR. x40.

In the lung, virus antigen-laden histiocytic cells were randomly scattered within the submesothelial areas of the pleura, interstitial and perivascular areas as well as peribronchiolar sites (Fig 3.8). In addition, occasional spindle-shaped cells of perivascular location, most likely pericytes, contained small amounts of intracytoplasmic antigen.

Figure 3.9: An immunohistochemically stained section of lung taken from a BEFV- affected steer 1 week after cessation of viraemia. Note the intracytoplasmic viral antigens within round cells presumed to be macrophages. The fresh spleen, haemal node, lymph node, and synovial fluid of this animal obtained during necropsy examination also tested positive for BEFV by qRT-PCR.x40.

In the heart, low numbers of antigen-laden macrophages were multifocally distributed within areas of focally degenerate myocytes and rarely within interstitial connective tissue (data not shown). In skeletal muscle, very rare numbers of macrophages and spindle cells of perivascular location that contained variable amounts of intracytoplasmic viral antigen were variably present. In addition, extremely low numbers of spindle-shaped and histiocytic cells with intracytoplasmic

78 viral antigen were seen in the glomerular and perivascular sites in the kidney section and urinary bladderof one animal. In the animal euthanased at 23 days, there was occasional intracytoplasmic viral antigen within a few neutrophils and macrophages located in areas of moderate proliferative lymphoplasmacytic synovitis (Figure 3.10).

Figure 3. 10: An immunohistochemically stained section of synovial membrane

(stifle joint) taken from a BEFV-affected heifer 23 days after cessation of viraemia.

Note few cells with intracytoplasmic viral antigen along with low-grade background staining. The fresh spleen, haemal node, and skeletal muscle of this animal obtained during necropsy examination tested positive for BEFV by qRT-PCR. ×100.

In the liver, few antigen-laden histiocytic cells as well as occasional spindle shaped cells of perivascular location were observed in the capsule and portal triads (Fig

3.12).

Figure 3.11: An immunohistochemically-stained section of the liver taken from a

BEFV-affected heifer 23 days after the initial BEF diagnosis. Note low numbers of cells within the subcapsular cells region that contain intracytoplasmic BEFV antigen.

Not seen in this photomicrograph are the occasional viral antigen-positive cells with histiocytic and spindle shape morphology that were present in the portal areas and perivascular sites. The fresh spleen, haemal node, and skeletal muscle of this animal obtained during necropsy examination tested positive for BEFV by qRT-PCR. ×40.

3.4.7 Transmission electron microscopy

Within an ultrathin section of a haemal node taken from the BEFV-infected case study steer euthanased within a week of being diagnosed with BEF, characteristic bullet-shaped (rhabdovirus) virus particles, measuring approximately 75 × 140 nm, were observed budding into smooth-sided vesicles within the cytoplasm of nucleated cells of presumed histiocytic morphology (data not included).

3.5 DISCUSSION

The major objectives of this study were to define tissue tropism of BEFV and to elucidate the potential replication sites of this virus in cattle. Based on IHC and qRT-

PCR data, tissue tropism of BEFV in cattle appears more extensive than previously known. Variable amounts of viral antigen were demonstrated by IHC in the spleen, haemal node, liver, lung, kidney, heart, skeletal muscle and unequivocal post- viraemic presence of BEFV was supported either by qRT-PCR in the spleen, haemal node, skeletal muscle and synovial fluids (A quantitative analysis was attempted after thesis review using Image J but the high background staining apparently resulting from the long storage of the slides negatively impacted the ability to discriminate mild positive staining and background staining). Moreover, visualisation of characteristic rhabdoviral virions was documented in the haemal node sections from one animal. These data represent the first study in which tissue tropism and potential replication sites of BEFV are evaluated through simultaneous demonstration of specific viral antigen and RNA in biological specimens taken from cattle naturally infected with BEFV. Considering the qRT-PCR data overall, spleen and haemal node were the organs most consistently found to be BEFV-positive, the longest time of viral RNA detection being 120 days following the initial BEF

81 diagnosis. These findings confirm that BEFV may localise in lymphoid tissues beyond the viraemic phase of the infection thus raising the possibility that the virus may replicate in the two lymphoid organs during and after cessation of viraemia.

However, since all specimens from the heifers euthanised at 30 and 150 days tested negative for viral RNA (Table 3.1), such post-viraemic localisation of BEFV in lymphoid tissues appears to vary between animals. This may be due to undefined host factors, with the quality of the immune response against BEFV playing a major role. For example, as the viral RNA was demonstrated in tissues at the time when the virus neutralising antibody response was peaking, the preferential virus localisation in primary lymphoid tissues may contribute to the immune stimulation that is required to sustain the long-lasting anti-BEFV antibody response typically seen in cattle. Unpublished data based on work done at BVL suggest that virus neutralising antibody titres continue to be detectable for several years in cattle following natural exposure to BEFV.

It should be noted that most of the BEFV antigen was distributed within the cytoplasm of cells with a morphology consistent with macrophages, dendritic cell-like cells, and probable pericytes. While definitive identification of the antigen-laden cells would have been worthwhile, immunophenotyping attempts were unsuccessfull most probably due to antigen denaturation since the tests were done after the tissue specimens had been in formalin for a long time. It is also noteworthy that in the present study, no virus antigen was evident within the mesothelial cells of the epicardium, lung, spleen or liver. Previously, infection of mesothelial cells with BEFV was instead reported by Young and Spradbrow (1985) in different thoracic and abdominal organs. Since qRT-PCR performed on peritoneal and other body fluids

82 was positive for BEFV in two heifers with acute BEF, it is likely that in association with primary polyserositis that usually occurs during acute BEF, inflammatory cells loaded with viral antigen may colonise the serosal surfaces and shed the virus into the body cavity fluids. Ultimately, the adsorbed viral particles may be taken up by submesothelial dendritic cells and macrophages as part of the innate immune response paving the way to antigen processing and presentation thus culminating into the initiation and progression of the adaptive immune response. Since the IHC assay is superior to the fluorescent antibody test in terms of histological localisation of microbial antigens in tissue specimens, it is possible that the viral antigen described by Young and Spradbrow (1985) within mesothelial cells may have been present within the various cells that, in the present study, were clearly infiltrating the submesothelial areas of different visceral organs. The intrahistiocytic viral antigen seen in the present study might reflect the phagocytic role of these cells, although a possible role of these cells in the replication and/or dissemination of viral particles

(Krakowka et al. 2001; Maclachlan et al., 2009) cannot be ruled out. For example, porcine circovirus-2 replicates within and is disseminated by monocytes and macrophages and a similar role has been demonstrated for monocytes and macrophages during BTV infection in ruminants (Maclachlan et al., 2009), and for dendritic cells in Nipah virus-infected animals (Mathieu et al., 2011). In this study, viral antigen has also been detected in neutrophils, as previously reported, (Burgess and Spradbrow, 1977; Theodoritis, 1969; Young and Spradbrow, 1985), thus supporting a possible phagocytic role during the infection.

In a comprehensive study on the pathogenesis of BEF by Basson et al. (1970), the authors reported pericyte hyperplasia as a consistent feature of this infection.

Interestingly, the role of pericytes as potential replication sites of a number of other viruses has also been reported (Alcendor et al., 2012; Chen et al., 2014; Nakagawa et al., 2012). In the study reported by Alcendor et al. (2012), the replication of cytomegalovirus within human pericytes of the brain was documented. Likewise, infection of pericytes by HIV-1 (Nakagawa et al., 2012), and Japanese encephalitis virus (Chen et al., 2014) has been reported. The consistent immunostaining of

BEFV antigen seen in spindle-shaped cells with a perivascular location in the present study suggests that pericytes and/or related myoepithelial cells, along with phagocytes, are likely to be potential replication sites for BEFV in cattle. Whether the presumed pericytes were hyperplastic or not could not be definitely assessed during the present study as immunophenotyping for these cells was not attempted due to lack of a suitable primary antibody probe.

It is important to note that eight out of nine animals had already seroconverted by the time the post-mortem examinations were performed. Therefore, it is likely that humoral and cellular immune mechanisms would have been underway by the time necropsy examination was conducted. This is further supported by the prevalent distribution of viral antigen-containing histiocyte and/or dendritic cell-like cells within the lymphoid follicles of spleen, haemal node, and lymph node along with occasional cells that were seen towards the centre of the germinal centres. Like in other mammalian species, initiation and progression of the adaptive cellular immune processes in domestic ruminants primarily take place within secondary lymphoid organs including spleen, haemal node and lymph node (Schultz et al., 1973; Pega et al., 2013). Therefore, it would be expected that dendritic and other antigen processing and presenting cells such as macrophages may translocate viral antigen

84 from peripheral tissue sites towards different lymphoid organs as part of the adaptive immune response. The homing of antigen-laden dendritic cells and other immune cells into secondary lymphoid organs is a widely accepted phenomenon in mammalian immunobiology (Bajenoff et al., 2003; Creusot et al., 2009; Zhang et al.,

2013). Taken together, all the present IHC data suggest that the preferential homing of antigen-laden histiocytic and dendritic cells occurs during BEFV infection. This immunological phenomenon is mainly attributed to chemokine and cytokine- mediated receptor and ligand molecule expression by antigen presenting cells and high endothelial venules located within secondary lymphoid organs (Bajenoff et al.,

2003; Zhang et al., 2013). Of the three lymphoid organs evaluated in this study, the haemal node appeared to have a higher number of presumed intrafollicular dendritic cells and histiocytes with intracellular viral antigen than the spleen or lymph node.

Moreover, as both the haemal node and spleen tested qRT-PCR-positive for viral

RNA in a number of animals, it is possible that the former may express a higher density of homing receptors for dendritic cells, macrophages, and other antigen presenting cells than the spleen or lymph node. Previous studies have reported the important contribution of the haemal node in the immune processes of domestic ruminants (Ezeasor et al., 1989; Zhang et al., 2012). The present data therefore suggest that together with the spleen and lymph node, the haemal node is likely to be an important site for generation of immune responses as well a potential replication site for the BEFV in cattle. This hypothesis was further supported by the ultrastructural observation of rhabdoviral virions in a section of haemal node taken from a steer 6 days after cessation of viraemia.

3.6 CONCLUSIONS

In conclusion, successful evaluation of the tropism of BEFV for cattle tissues was achieved through the application of IHC, qRT-PCR, and virus isolation techniques on post-mortem and other specimens derived from cattle naturally infected with BEFV.

Besides, the present research data also elucidate on the likely tissue replication sites of the BEFV in cattle. On the basis of the present findings, it is likely that BEFV may transiently localise and possibly replicate in lymphoid tissues beyond the viraemic phase of BEFV infections in cattle. As such, there is justification for post-mortem sampling of the spleen and haemal node especially in cases where other biological samples are not available to provide a reliable retrospective diagnosis in animals dying from suspected BEF. From a biosecurity point of view, however, it is highly unlikely that presence of viral antigens and RNA in meat may pose a concern as

BEFV RNA is rapidly inactivated by low pH prevailing in skeletal muscle during the post-slaughter period.

CHAPTER 4

VIRAL NEUROTROPISM, PERIPHERAL NEUROPATHY AND OTHER

MORPHOLOGICAL ABNORMALITIES IN BOVINE EPHEMERAL FEVER VIRUS-

INFECTED DOWNER CATTLE

This chapter is based on data already published as a full research article in the

Australian Veterinary Journal. For consistency of formatting within the thesis and to minimise self-plagiarism concerns, modifications have been made to the already published text.

1. Research paper published in the Australian Veterinary Journal Barigye, R1,2,3, Davis, S1, Hunt, R1, Hunt, N1, Walsh, S1, Elliott, N1, Burnup, C1,

Aumann, S1, Day, C1, Dyrting, K1, Weir, R1, Melville, L.F1. 2016. Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle. Australian Veterinary Journal, 2016, Vol 94, pp.

362-370.

1Berrimah Veterinary Laboratories, Department of Primary Industry and Resources,

GPO Box 3000, Darwin, NT 0801, Australia;

2School of Veterinary Science, University of Queensland, Gatton QLD 4343,

Australia;

3Department of Veterinary Medicine, College of Food and Agriculture, United Arab

Emirates University, PO Box 15551, Al Ain, UAE.

4.1 ABSTRACT

This study assessed the neurotropism of BEFV and described histomorphological abnormalities of the brain, spinal cord, and peripheral nerves that may causally contribute to paresis or paralysis in BEF. Four paralysed and six asymptomatic but virus-infected cattle were monitored, and blood and serum samples screened by qRT-PCR, virus isolation, and neutralisation tests. Fresh brain, spinal cord, peripheral nerve, and other tissues were qRT-PCR-tested for viral RNA, while the formalin-fixed specimens routinely processed for histology and immunohistochemistry in order to evaluate histomorphological abnormalities and viral antigen distribution. The neurotropism of BEFV was immunohistochemically confirmed in the brain and peripheral nerves and peripheral neuropathy was demonstrated in three paralysed but not the six asymptomatic but virus-infected animals. Wallerian degeneration (WD) was present in the ventral funicular white matter of the lumbar spinal cord of one paralysed steer and in the cervical and thoracic spinal cord segments of three paralysed animals. While no spinal cord lesions were seen in the BEFV-infected downer steer euthanased 6 days after cessation of viraemia, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. The present research data confirms neurotropism of BEFV in cattle and documents histomorphological abnormalities in peripheral nerves and brain, which along with spinal cord lesions may contribute to chronic paralysis in BEFV-infected downer cattle.

4.2 INTRODUCTION

A number of studies have previously indicated that inflammation and fever are hallmark features of BEF (Basson et al., 1970; Burgess and Spradbrow, 1977; St

George et al., 1984; St George et al., 1986; Uren et al., 1989). While these findings highlight the inflammation as a pivotal mechanism in the pathogenesis of BEF, the mechanistic events that underlie the paresis and chronic paralysis seen in some field

BEF cases remain a subject of scientific speculation. Based on current evidences, hypocalcaemia may contribute to neuromuscular weakness and therefore paresis/paralysis in infectedanimals (St George et al., 1986). This hypocalcaemia is easily reversed by parenteral infusion of calcium borogluconate (Nandi and Negi,

1999; St George et al., 1989). However, the extent to which inflammation contributes to paralysis/paresis in acute BEF is not clear. In the mid-1970s, fatal encephalitis was experimentally induced by intracerebral inoculation of a virulent

BEFV strain into two seronegative calves but not in an age-matched animal that was seropositive for maternal virus neutralising antibodies (Tzipori, 1975). Subsequently, severe bilaterally symmetrical Wallerian degeneration (WD) was demonstrated in spinal cord sections of four BEFV-infected animals and the authors of that work speculated that compressive myelopathy was the likely cause of the lesions (Hill and

Schulz, 1977). In three of the four animals reported in that study, WD was present in the first segment of the cervical spinal cord, while in the remaining animal it was localised in the lumbar spinal cord.

In the context of the pathogenesis of the chronic paralysis seen in normocalcaemic

BEFV-infected animals, it is therefore important that the neurotropism and neuropathogenicity of BEFV is thoroughly investigated.

The present study therefore assessed the potential neurotropism of BEFV in adult cattle naturally infected with the virus, and described the different histological abnormalities that could be causally linked to chronic paralysis in affected animals.

4.3 MATERIALS AND METHODS

4.3.1 Study animals and clinical monitoring

The present case control study was based on 10 adult cattle selected on the basis of confirmed BEFV infection, as well as negative control tissues from BEFV-negative animals. All the 10 cattle were raised at research farms owned by the DPIF, NT

Government, Australia. Following natural BEFV exposure and clinical sickness, the animals were monitored for neurological and other symptoms. Video footage and still photographs taken in two of the four paralysed animals were analysed, and where available, the clinicians’ records reviewed for the nature of neurological signs. Of the four neurological cattle, animals No. 3 and 4 presented with paralysis from around the time of the BEF diagnosis until when euthanasia and necropsy examinations were done, i.e.7 and 23 days after the initial BEF diagnosis, respectively. On the other hand, animal No. 6 demonstrated total recumbency and paralysis that lasted until the time of necropsy examination 42 days later. Animal No. 8 initially presented with severe paralysis and recumbency for four days and then continued to show residual proprioceptive deficits and mild ataxia until the day of necropsy examination

93 days later.

4.3.2 qRT-PCR for BEFV

Samples of peripheral blood, fresh tissues as well as pericardial, thoracic, peritoneal and synovial fluids were tested for BEFV by qRT-PCR using a protocol originally

90 standardised by Lew et al. (2006) and described in section 2.3.2. The primers and probes used in this study have also been listed in Table 2.1 above and detailed information pertaining to the negative controls presented under appendix 3.

4.3.3 Virus neutralisation test and virus isolation

The virus neutralisation test was done on heat-denatured test serum samples using a modified version of the protocol initially reported by Uren et al. (1994) and described in section 2.3.3 above. Virus isolation from blood samples was also performed following the protocol as described in section 3.3.2. For the isolation of

BEFV from tissue samples, the specimens were macerated and mechanically agitated to yield fine, cell-rich slurries. The latter were then mixed with a small amount of 0.12% trypsin (SAFC, Sigma-Aldrich, St Louis, MO, USA), homogenised by additional mechanical agitation, and then centrifuged at 4000 RPM for 10 min.

The supernatants were harvested and inoculated onto monolayers of C6/36 Aedes albopictus (mosquito/insect) cells and the rest of the virus isolation in BSR cells done as described in section 3.3.2.

4.3.4 Necropsy examination of BEFV-infected cattle

All the four BEFV-infected and paretic/paralysed animals were humanely euthanised

7, 23, 42, and 93 days after the initial confirmatory BEF diagnosis. The five non- paralytic BEFV-infectedanimals were euthanised at 1, 30, 60, 120, and 150 days, and one animal died from the natural disease one day after the onset of clinical signs

(It should be noted that these are the same animals on which the study described under chapter 3 was based). Prior to necropsy examination, the sick animals were evaluated for clinical signs, bled, and then euthanised by intravenous infusion of

Lethabarb per ethical guidelines (Animal Ethics Committee Permit No. A12028). With the exception of two animals in which peripheral nerve tissues were not collected, all organ systems including the peripheral nerves, spinal cord, and brain were examined for macroscopical abnormalities and fresh and formalin-fixed tissue and other biological specimens collected. In seven out of the 10 animals including two variably paralysed steers, specimens of the left and right gluteal nerves as well as hamstring muscles were collected. Peripheral nerve tissues were not collected from the heifer that died from acute BEF (animal No. 2), only brain and spinal cord were collected from the paralysed steer (animal No. 6), and a comprehensive specimen collection was done in animal No. 3 (Table 4.1). The specimens collected from animal No. 3 included peripheral nerve tissue from the left and right brachial plexuses, left and right gluteal nerves, left and right fibular nerves, the two cerebral hemispheres, thalamus, mid-brain, cerebellum, medulla oblongata, and several skeletal muscle groups (Table 4.1). In addition, negative control neural tissues were collected from a

BEFV-negative steer (brain) and calf (peripheral nerve tissue).

4.3.5 Tissue processing and histological evaluation

The formalin-fixed tissues were routinely processed, paraffin embedded, and the 4

μm sections deparaffinised and stained with haematoxylin and eosin. The H&E- stained sections of spinal cord segments (including cervical spinal cord, thoracic spinal cord, and lumbar spinal cord segments), cerebrum, cerebellum and medulla oblongata were examined for neuropathological abnormalities. Neurodegenerative or inflammatory changes were subjectively scored as + (mild), ++ (moderate), or +++

(severe). Sections of the peripheral nerves were evaluated for the presence of microscopical lesions. To further characterise the peripheral nerve lesions, sections

92 of the affected nerves were also stained with Alcian blue pH 2.5, Luxol fast blue,

Periodic Acid Schiff (PAS), and Von Kossa stains. Histological evaluation was done using a light microscope (Olympus-BX41, Model U-MDOB3) and photomicrographs taken with the Olympus Labsens imaging software.

4.3.6 IHC test for BEFV antigen

The paraffin-embedded tissue blocks along with the negative control nerve and brain tissues were trimmed at 4 µm and duplicate sections adhered onto salinised

(‘positively charged’) slides. The IHC positive control was a sample of spleen taken from a Droughtmaster bull that was the first animal to test positive for BEFV RNA by qRT-PCR. Note that a section of the medulla oblongata from a BEFV-negative steer that died from listeriosis, peripheral nerve tissue from a BEFV-negative calf, and peripheral nerve tissue from an adult sheep were used as the negative controls for the IHC assay performed on nervous tissues. The rest of the IHC protocol was performed as described in section 3.3.7.

4.4 RESULTS

4.4.1 Animals and clinical monitoring

From the time animal No. 3 was found viraemic and sick, the Brahman steer remained paretic/paralysed and recumbent until the time of necropsy examination seven days later. The clinical signs included recumbency, fever, anorexia, serous discharges, paresis, as well as aggressiveness and futile attempts to stand up when approached. Animal No. 4 was a paretic/paralysed Brahman heifer that showed recumbency and progressive paralysis from the initial BEF diagnosis until necropsy examination 23 days later. The other paretic/paralysed animal (No. 8) was a

Droughtmaster steer in which the clinical signs documented during the first four days of sickness included somnolence, anorexia, muscle fasciculations, proprioceptive deficits, and loss of extensor muscle tone. This animal was totally recumbent for four days, reluctant to move for additional five days, appeared disoriented, and exhibited an unsteady gait and partial blindness. Residual neurological deficits that included mis-stepping of the foreleg and mild ataxia were still apparent at the time of euthanasia and post-mortem examination 93 days later. When found, the fourth paralysed Brahman steer (animal No. 6) demonstrated recumbency and inability to stand despite good appetite and appearing bright. To some extent, the animal could bear weight on the front but not the hind limbs and euthanasia and post-mortem examination were elected six weeks after the original BEF diagnosis. Obvious neurological signs and recumbency were not observed in the other six BEFV- infected animals euthanased at different time points following the initial BEF diagnosis.

4.4.2 qRT-PCR results

With the exception of the paralysed Brahman (No. 6) and Droughtmaster (No. 8) steers, a qRT-PCR test on blood at the time of the initial BEF diagnosis was positive in the other 8 of the10 animals (Table 4.1). By the time of necropsy examination, however, only animal No. 1 euthanised one day after the initial diagnosis and animal

No.2 which died from the natural disease were still viraemic (Table 4.1). With the exception of the spinal cord, which was not tested by qRT-PCR, all the other tissues and body fluids were positive for viral RNA in animal No. 1. In animal No. 2, the specimens that tested positive for viral RNA included neural tissues (peripheral nerve, spinal cord, plus cerebrum, cerebellum and medulla oblongata) as well as

94 peripheral blood, synovial fluids from stifle and hock joints, as well as pericardial, thoracic, and peritoneal fluids, spleen, haemal node, liver, lung, kidney, urinary bladder, heart, skeletal muscle, tongue, and synovial membrane (Table 4.1).

Regarding animal No. 3, the spleen, haemal node, lymph node, and synovial fluids from the two stifle joints were positive for viral RNA (Table 4.1). In animal No. 4, only the spleen, haemal node, and skeletal muscle were positive for viral RNA and in animal No. 8, only the spleen tested positive (note that haemal node was not tested)

(Table 4.1). In the three animals euthanised at 60 (No. 7), 93 (No. 8) and 120 (No. 9) days, the spleen, the spleen and haemal node, and only the haemal node tested positive for viral RNA, respectively (Table 4.1). Interestingly, none of the tissues, peripheral blood, or body fluids taken from animals No. 5 and 10, euthanised respectively at 30 and 150 days following the initial BEF diagnosis tested positive for viral RNA (Table 4.1).

4.4.3 Virus neutralisation test and virus isolation results

At the time of the initial BEF diagnosis, 8/10 of the virus-infected animals had negative virus neutralising antibody titres. With the exception of animal No. 6 in which serum was not tested, the virus neutralising antibody titres at the time of necropsy were positive in the other seven animals (Table 4.1). Notably, BEFV was isolated from blood of the two animals at the time of the initial BEF diagnosis, and from blood, spleen, haemal node, and lymph node of the heifer that died from acute

BEF. However, no virus was isolated from blood, spleen, haemal node and skeletal muscle of the heifer that was euthanised at 23 days despite these tissues testing positive for viral RNA. Interestingly, BEFV was successfully isolated from the spleen

95 of the non-viraemic steer that was euthanised one week following the initial BEF diagnosis.

Table 4.1. A summary of the data from the neuropathology study of BEFV-infected cattle.

Ani Days qRT-PCR test qRT-PCR qRT-PCR Virus Microscopic Post-mortem specimens that were mal elapsed on peripheral test on test on neutralising lesions in neural positive by blood during peripheral nerve antibody titre at tissues for BEFV by the qRT-PCR test necropsy acute BEF blood on day tissue time since of necropsy of necropsy initial exam BEF diagnosis

1 Euthanased 1 + + + 48 None All other tissues; All other body fluids Died from aPNo 2 acute BEF 1 + + + <4.5 All other tissues; All body fluids PNN (+++) 3 Total 7 + - - 160 Spleen, haemal node, lymph node, paralysis synovial fluid Total PNN (++ to +++) Spleen, 4 paralysis, 23 + - - 144 CSC (+ to ++) haemal node, progressive TCC (+ to ++) skeletal muscle LSC (+ to ++) Not 5 paralysed 30 + - - <4.5 None None Total 6 recumbency, 42 - - NE NE bCSC WD (++) Tissues not tested chronic TSC (++) paralysis Not None 7 paralysed 60 + - - 48 Spleen, haemal node 4 days PNN (+) 8 paralysis & 93 - - - 72 CSC WD (++) Spleen (haemal node not tested) recumbency, TCC (+) residual LSC (+) ataxia Not 9 paralysed 120 + - - 32 None Haemal node

Not 10 paralysed 150 + - - 40 None None

Legend: aPNo = Peripheral nerve tissue not collected; bOnly brain and spinal cord tissues were collected. In addition to peripheral blood, and peritoneal, pericardial and synovial fluids, peripheral nerve, spinal cord and brain were tested by qRT-PCR. Note that a virus-neutralising antibody titre <4.5 is considered negative. H&E-stained sections of spinal cord segments, cerebrum, cerebellum and medulla oblongata were examined for neuropathological abnormalities. When sighted, neurodegenerative or inflammatory changes were subjectively scored as + (mild), ++ (moderate), or +++ (severe). BEFV, bovine ephemeral fever virus, CSC, cervical spinal cord; TSC, thoracic spinal cord; LSC, lumbar spinal cord; NE, not examined; PNN, peripheral nerve neuropathy; WD,

Wallerian degeneration.

Table 4.2: A summary of the histopathology data that were obtained from the neuropathology study of BEFV-infected cattle.

Animal ID

1 2 3* 4* 5 6* 7 8* 9 10

Number of days elapsed from BEF diagnosis and necropsy examination

1 1 7 23 30 42 60 93 120 150

Peripheral nerve

(neuropathy) NE - +++ ++ - NE - + - -

Cervical spinal cord

(Wallerian degeneration) NE - - ++ - ++ - ++ - -

Thoracic spinal cord

(Wallerian degeneration) NE - - + - +++ - + - -

Lumbar spinal cord

(Wallerian degeneration) NE - - + - - - + - -

Cerebrum

(Perivascular lymphocytic encephalitis) + ------+ - -

Cerebellum

(Perivascular lymphocytic encephalitis) ------+

Medulla oblongata

(Perivascular lymphocytic encephalitis) - - - - + + + + - -

Legend: H&E-stained sections of spinal cord segments, cerebrum, cerebellum and medulla oblongata were examined for neuropathological abnormalities. When sighted, neurodegenerative or inflammatory changes were subjectively scored as + (mild),

++ (moderate) or +++ (severe). The asterisk denotes identification numbers of the four animals that demonstrated chronic paralysis following acute BEFV infection. BEFV, bovine ephemeral fever virus; NE, not examined.

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4.4.4 Neuropathological and other pathological abnormalities

Only histomorphological abnormalities potentially affecting locomotion and contributing to the neurological symptoms are presented in Tables 4.1 and Table 4.2 and the text below. These include abnormalities of peripheral nerves, spinal cord, brain, skeletal muscle, and synovial membrane of the stifle joints. Notably, no lesions were seen in peripheral nerve and spinal cord sections of the six BEFV-infected but otherwise asymptomatic cattle (animals No. 1, 2, 5, 7, 9 and 10) (Table 4.1; Table

4.2). In three of the four paretic/paralytic BEFV-infected animals, sections of peripheral nerves demonstrated degenerative changes morphologically consistent with peripheral neuropathy (Figures 4.1, 4.2, and 4.3; Table 4.2). Most severe in animal No. 3, moderate in the animal No. 4, and mild in animal No. 8, these lesions were characterised by focally extensive areas of intrafascicular degeneration and necrosis consisting of loosely arranged myxomatous debris in which there were sparsely distributed nucleated cells interpreted to be macrophages and/or pyknotic glial cells (Figures 4.1, 4.2, and 4.3; Table 4.2). In addition, the degenerate nerve tissue debris was Alcian blue-reactive (Figure 4.2) and remarkably stained negative for myelin (Figure 4.3) when compared to the healthy nerve in the BEFV-infected but asymptomatic animal (Figure 4.4). Moreover, the degenerate nerve tissue debris stained negative with PAS and Von Kossa stains (data not included). In animal No. 3 used for the case study, the lesions were more pronounced in the peripheral nerve taken from the right brachial plexus followed by the left brachial plexus, and less marked in sections of the gluteal and fibular nerves. In this animal, there was also a moderately proliferative neutrophil-rich lymphoplasmacytic synovitis involving the left and right stifle joints and characterised by a perivascular pattern along with occasional lymphoid nodule formation. This animal also demonstrated multifocal

101 areas of very low-grade focal myopathy associated with mild myocyte disruption and variable numbers of infiltrating histiocytes. The rare focal myodegenerative lesions were present in the right longismuss dorsii, right/left biseps branchii, left semitendinosus, right supraspinatus, and left gluteus medius muscles. In animal No.

4, the gluteal nerve also showed mild degenerative lesions along with mild to moderate WD that predominantly involved the ventral funiculi of the cervical, thoracic, and lumbar spinal cord segments (Table 4.1 and 4.2). Very mild perivascular lymphoplasmacytic meningitis (data not shown) was also demonstrated in the spinal cord dura of all the seven BEFV-infected animals. In the only section of skeletal muscle taken from the hamstring area of animal No. 4, there were rare foci of myocyte degeneration associated with loss of cross-striations, and low-grade lymphohistiocytic cell infiltrations (data not shown). In animal No. 8, there was mild to moderate symmetrical WD that was particularly pronounced in the ventral funiculi of the cervical spinal cord white matter and less marked in the thoracic and lumbar spinal cord. This animal also demonstrated mild to moderate perivascular lymphocytic meningoencephalitis in the cerebrum and medulla oblongata (Table

4.2). In animal No. 6, one section of medulla oblongata demonstrated a low-grade to moderate perivascular lymphoplasmacytic encephalitis (data not shown) and mild to moderate WD was seen in the ventral funiculi of the cervical and thoracic spinal cord segments (Table 4.1). The most relevant morphological diagnoses in animal No. 1 included mild perivascular lymphocytic encephalitis in the cerebrum, and mild lymphocytic and neutrophilic synovitis affecting the right carpal joint. In animal No. 5, there was very mild perivascular lymphoplasmacytic meningitis in the thoracic and lumbar spinal cord dura (data not shown) as well as very mild diffuse neutrophilic synovitis in the left and right stifle joints. In animal No. 7, there was very mild

102 lymphocytic perivascular encephalitis in the medulla oblongata and mild perivascular lymphoplasmacytic meningitis in the thoracic spinal cord dura. In animal No. 9, there was mild lymphocytic meningitis in the thoracic spinal cord segment, and mild neutrophilic/lymphocytic synovitis with a predominant perivascular pattern. In animal

No. 10, there was mild lymphoplasmacytic to neutrophilic synovitis in the left stifle joint and low-grade perivascular lymphoplasmacytic inflammation involving a few small blood vessels within the cervical and thoracic spinal cord dura. Very mild perivascular encephalitis was also present in the section of the cerebellum.

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Figure 4.1: Peripheral nerve taken from the left brachial plexus of a chronically paralysed and recumbent BEFV-infected steer (animal no. 3) euthanised within 6 days of the initial BEF diagnosis. Note the focally extensive areas of myxomatous degeneration surrounding the normal portions of the nerve, H&E, ×40.

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Figure 4.2: Peripheral nerve taken from the left brachial plexus of a chronically paralysed and recumbent BEFV-infected steer (animal no. 3) euthanised within 6 days of the initial BEF diagnosis. Note the abundant Alcian blue-reactive and light blue staining myxomatous material surrounding remnants of nerve fibre tissue

(Alcian blue, pH 2.5), ×20.

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Figure 4.3: Peripheral nerve taken from the left brachial plexus of a chronically paralysed and recumbent BEFV-infected steer (animal no. 3) euthanised within six days from the initial BEF diagnosis. Note the extensive pauci-myelinated areas within the nerve tissue. Punctate myelin staining is still visible in the relatively intact areas of nerve (Luxol fast blue), ×40.

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Figure 4.4: Section of the left gluteal nerve of a BEFV-infected but asymptomatic heifer (animal no. 10) showing abundant myelin staining throughout the nerve tissue

(Luxol fast blue), ×40.

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4.4.5 IHC test on nervous tissues

Regarding neurotropism of BEFV, very rare cells presumed to be Schwann cells

(Figure 4.5) and spindle-shaped cells of perineurial and epineurial connective tissue location demonstrated small amounts of intracytoplasmic viral antigen in the BEFV- infected cattle. Notably, the BEFV antigen-positive presumed Schwann cells were consistently demonstrated in sections of peripheral nerves of 9/10 of the BEFV- infectedanimals (Figure 4.5) but not in the negative control (Figure 4.6) nerve tissues. In addition, sections of peripheral nerve from the BEFV-negative bovine used as a negative control did not show any immunoreactivity (Figure 4.7). Finally, there was no correlation between presence of the viral antigen and degenerative lesions in the nerves (Figure 4.8). In animal No. 6 - one of the four paralysed cattle - the medulla oblongata showed abundant intrahistiocytic viral antigen distributed within perivascular sites and neuropil (Figure 4.9). Note that a corresponding medulla oblongata section probed with the negative mouse serum (Figure 4.10) and another from a BEFV-negative animal that died from neurological listeriosis (Figure

4.11) did not show any viral antigen. In addition to the medulla oblongata, the choroid plexus in the brain section from animal No. 6 also showed a few virus- antigen-laden macrophages that were innocuously scattered within the perivascular sites.

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Figure 4.5: A. Section of the left gluteal nerve from a BEFV-infected heifer (animal no. 4) euthanised 23 days from the initial BEF diagnosis. Note few Schwann cells with abundant intracytoplasmic positive stain. The section was stained with the mAb DB5 and counterstained with Mayer’s haematoxylin, ×40; B. Left gluteal nerve from a BEFV-infected heifer (animal no. 4) euthanised 23 days from the initial BEF diagnosis. Note the absence of positive staining. The section was probed with negative control mouse serum and counterstained with Mayer’s haematoxylin, ×40.

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Figure 4.6: Gluteal nerve from a BEFV-negative animal probed with the mAb DB5 and counterstained with Mayer’s haematoxylin. Note the absence of BEFV antigen,

×40.

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Figure 4.7: Peripheral nerve taken from the right brachial plexus of a chronically paralysed and recumbent BEFV-infected steer (animal no. 3) euthanised within six days from the initial BEF diagnosis. Note the absence of immunostaining within and outside areas of nerve tissue degeneration and necrosis. The section was probed with the mAb DB5 and counterstained with Mayer’s haematoxylin, ×40.

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Figure 4.8: A. Section of medulla oblongata from a neurological BEFV-infected steer (animal no. 6) euthanised 42 days from the initial BEF diagnosis. Note few macrophages with abundant intracytoplasmic staining forming associated with mild lymphoplasmacytic encephalitis. The section was probed with the mAb DB5 and counterstained with Mayer’s haematoxylin, ×40. B.

Section of medulla oblongata from a BEFV-infected neurological steer (animal no. 6) euthanised 42 days from the initial BEF diagnosis. Note the absence of immunostaining for viral antigens. The section was probed with negative control mouse serum and counterstained with Mayer’s haematoxylin, ×40.

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Figure 4.9. Section of the medulla oblongata from a BEFV-negative steer that died from listeriosis, probed with the mAb DB5 and counterstained with Mayer’s haematoxylin. Note the absence of BEFV antigen. BEFV, bovine ephemeral fever virus; mAb, monoclonal antibody, ×40.

4.5 DISCUSSION

The two major objectives of this research were to investigate the neurotropism of

BEFV in cattle and to describe and characterise any neuropathological lesions that may contribute to the chronic paralysis seen in a minority of BEFV-infected animals.

Regarding the first objective, the present research data provided evidence consistent

113 with neurotropism of BEFV in both central and peripheral nervous system of cattle.

Peripheral nerve tissue, spinal cord, and brain of acutely infected animals were positive for viral RNA thus supporting the hypothesis that BEFV infects these tissues.

This most likely occurs in viraemic animals through the blood system. While post- viraemic neural tissues were qRT-PCR-negative, viral antigens were consistently demonstrated within brain stem lesions of one paralysed steer and within a few presumed Schwann cells of some peripheral nerves and spindle-shaped cells with a perineurial and epineurial location. However, it is still uncertain whether the virus gains entry into neural tissues via blood or through neuromuscular junctions like is the case for rabies virus infection. Neurotropism of the rabies virus is widely known and its affinity for Schwann cells and other peripheral nerve structures is well- documented (Lycke and Tsiang, 1987). With this viral infection, neurotropism has been considered as a part of the axoplasmic anterograde transport mechanism that conveys the rabies virus towards the spinal cord and brain (Castellanos et al., 2000;

Lycke and Tsiang, 1987). Since BEFV has a viraemic phase and therefore may not necessarily require the peripheral nerve route to get to the brain, the affinity for

Schwann cells may instead play a role in the transient persistence within the nervous system as reported for a number of other neurotropic viruses like rabies, herpes, mumps viruses (Love et al., 1987; Lycke et al., 1988; Lycke and Tsiang, 1987).

Strikingly, and reminiscent of rabies pathogenesis (Castellanos et al., 2000; Lycke and Tsiang, 1987), BEFV antigen was also seen in presumed Schwann cells of peripheral nerves embedded in the lingual musculature. Since dissemination of

BEFV during the viraemic phase is via blood (Mackerras et al., 1940; Theoriditis,

1969), the evolutionary advantage of BEFV innocuously persisting within the peripheral nerves is not clear. Further to this, the present IHC data do not show

114 spatial localisation of viral antigen within areas of damaged peripheral nerve tissue.

This further suggests that the presence of BEFV within peripheral nerves, or an antigenic component thereof, may be indicative of passive intraneural virus presence rather than being a direct cause of peripheral neuropathology. However, it should be noted that low-grade perivascular inflammation was seen around small blood vessels of the spinal cord dural membranes. Since this was also seen in non-neurological

BEFV-infected animals, the significance of the mild dural lesion in the spinal cord is not clear.

Regarding the second study objective, peripheral nerve, spinal cord, and brain tissues from study animals were carefully examined for histopathological changes and virus antigen distribution. Interestingly, abundant intrahistiocytic viral antigen was observed alongside low-grade lymphoplasmacytic infiltrates within perivascular and intra-neuropil sites of the medulla oblongata in a chronically paralysed BEFV- infected steer. The nature and severity of the brain stem lesions strongly suggest a causal relationship between viral antigen presence and chronic paralysis in this animal. In animal No. 3, the severe neuropathy was clearly more pronounced in the nerve specimen from the brachial plexuses when compared to the gluteal and fibular nerves. Since there was no spatial correlation between viral antigens and nerve tissue damage, and considering the morphological features of the lesion, compression and/or ischaemic neuropathy were interpreted to be the most likely factors underlying this nerve lesion. It is possible that the compressive forces responsible for the degenerative peripheral nerve lesions were stronger in the anterior than in the posterior portion of the animal. Compression neuropathy has been extensively studied in human medicine (Sunderland et al., 1976; Thatte et al.,

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2011) as well as in animal models (Dellon and Mackinnon, 1991; Rydevik et al.,

1981) with several authors speculating that this lesion is the primary cause of chronic paralysis in downer cattle (Aiello, 2016). According to the latter reference, prolonged recumbency may decrease lymphatic and venous drainage of the regional musculature with the building pressure ultimately causing compressive ischaemia of peripheral nerve tissue and muscle. In the animal euthanised after seven days of recumbency, there were several myodegenerative foci in various muscle groups along with fat necrosis in periarticular tissues of the stifle joints. On the other hand,

Hill and Schultz (1977) have suggested that chronic paralysis in some BEFV-infected downer cattle may be associated with WD occurring secondary to spinal cord trauma. Interestingly, WD was only seen in three out of the four paralysed animals in the present study, and the steer with the most severe peripheral neuropathy, but without spinal cord lesions, demonstrated the most severe paralysis. These findings may suggest that WD is not the only abnormality that may account for the BEF- associated chronic paralysis and that peripheral neuropathy may also be a significant contributory causal factor in the syndrome. Since the paralysed animal

(No. 3) did not have spinal cord lesions seven days after the onset of the clinical disease, spinal cord trauma probably arising from persistent efforts to get up over time as seen in video footage of two study steers may be the cause of the spinal cord WD as previously speculated by Hill and Schulz (1977). While these authors reported WD in cervical and lumbar spinal cord of two paralysed BEFV- infectedanimals, they did not examine specimens of peripheral nerves. The Alcian blue-positive but Luxol fast blue negative-myxomatous material within the affected nerves seen in the present study suggests the presence of intralesional acidic mucopolysaccharides. While similar neuropathology has not been documented in

116 cattle, focal mucoid degeneration was reported in a sural nerve lesion of a human patient (Meir and Bischoff, 1977). Pathological myelin breakdown may release several myelin-associated glycoproteins from damaged myelinated nerve fibres

(Filbin et al., 2003; Gendelman et al., 1985; Yiu et al., 2006). The acidic mucopolysaccharides that accumulated in the peripheral nerves of the BEFV- infected animals along with the paucity of myelin staining within the degenerate nerve tissue may suggest that formation of mucopolysaccharide-rich intermediates derived from the breakdown of myelin-sheath–associated-proteins occurred in these animals (Meir and Bischoff, 1977). On the other hand, Biasibetti et al. (2016) have reported age-related axonal degeneration and demyelination in bovine peripheral nerves derived from 10-20-year-old cattle but not in 12-29-months-old animals used as the negative control. Since the cattle used in the present study had a mean age of about 2-years-old, age-related peripheral neuropathy is unlikely. In summary, peripheral neuropathy detected in three of the four paralysed animals and WD seen in spinal cord sections of three of the four downers, as well as brain stem encephalitis seen in one of the BEFV-infected downers provide evidence for clinically relevant BEFV-associated neuropathology. The low-grade focal myodegenerative lesions seen in the paralysed animals were most likely a result of muscle trauma while the synovitis seen in some of the study animals was consistent with what previously reported in BEF (St George, 1986). None of the muscle or synovial lesions would have caused the level of chronic paralysis seen in the four downers. It should be noted that attempts to immunophenotype and definitively identify cells with intracyoplasmic viral antigen was unsuccessful. It is probable that this was due to antigen denaturation since the formalin tissue blocks had been stored for a long time by the time the IHC tests were attempted.

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4.6 CONCLUSIONS

In summary, the present research data provide evidence for viral neurotropism in peripheral nerves and the brain of BEFV-infected cattle. While the viral antigen was not associated with concurrent neuropathology in the peripheral nerves, the spatial localisation of the virus within areas of chronic inflammation in the brain stem of a chronically neurological steer has been demonstrated. In addition to WD in the spinal cord, peripheral neuropathy, as well as BEFV-induced brain stem encephalitis may constitute a spectrum of neuropathological abnormalities causally linked to chronic paralysis in a minority of BEFV-infectedanimals. It is apparent that the peripheral nerve lesions may constitute a component within a probable multifactorial pathogenesis of the downer cow syndrome in general. A concurrent study of BEFV- infected and non-infected downer cattle would aid in evaluating this hypothesis.

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CHAPTER 5

GENERAL DISCUSSION

5.1 The role of cytokines in the immunity and pathogenesis of BEF:

While a few publications havesuggested the potential role(s) played by proinflammatory cytokines in BEF pathogenesis (St George et al., 1995; Uren et al.,

1989), empirical studies have never been done to comprehensively confirm such hypothesis in adult cattle. In addition, while post-infection and post-vaccination immunity in BEF has thus far been attributed to virus neutralising antibodies (Aziz-

Boaron et al., 2013; Uren et al., 1994), the possible contributory role of cellular immunity, particularly the role of Th1 and Th2 cytokines, has never been exhaustively investigated. Of particular interest, the present study therefore sought to clarify the kinetics of specific cytokine networks that prevail and therefore may potentially mediate, at least in part, the innate-adaptive immune response transition in BEF. As such, the overall goal of the study was to elucidate the role of a selected number of cytokines in the pathogenesis and development of immunity against

BEFV infection in adult cattle. Specifically, the first part of this thesis is describing the significance of the data obtained from specific studies that focused on addressing two important research objectives namely:

1. Describing the plasma kinetics of proinflammatory cytokines (IL-1β, IL-6, and

TNF-α) alongside IL-10 (‘Th2 cytokine’ that is reported to be antagonistic to

the inflammatory and fever responses) during acute BEF. In part, this was

achieved by assessing how the cytokine expression kinetics were temporally

associated with the fever response in adult cattle naturally infected with

BEFV;

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2. Describing the kinetics of IL-2 and IFN-γ (‘Th1 indicator cytokines’), alongside

IL-6, and IL-10 (‘Th2 indicator cytokines’) during the innate-immune response

transition in adult cattle with BEFV infections. The second study objective was

specifically designed to indirectly elucidate on the potential contributory role of

cell-mediated mechanisms in the development of immunity against natural

BEFV infections in cattle through studying the kinetics of Th1 and Th2

cytokines during the BEF infection.

Under both study objectives, the relationship between the kinetics of the virus neutralising antibody response and the duration and resolution of viraemia was also assessed. After evaluating the data overall, it is apparent that the kinetics of expression of the cytokines as described above during acute BEF and the immediate post-viraemic period suggest potential roles played by these molecules both in the clinical expression of the disease (fever), and potentially in the development of immunity against the disease. Notably, there was a variable increase in plasma IL-

1β, IL-6, TNF-α, and IL-10 during natural BEFV infections with apparent upregulation of all but TNF-α preceding seroconversion. In addition, amongst the studied proinflammatory cytokines, IL-1β was the most strongly expressed and it appeared to correlate with the fever response in the first cohort of cattle under examination.

While the present data are interpreted with caution owing to the small sample size, and considering the anti-inflammatory properties of IL-10, it may be hypothesised that upregulation of IL-10 most likely plays a modulatory role that may potentially antagonise the fever response in animals acutely infected with BEFV. Moreover, as the increased plasma levels of IL-6 and IL-10 appeared to precede seroconversion, when the data are interpreted in the context of the literature, it may be inferred that the two cytokines may influence immunological events that pave way to B-cell

120 activation and seroconversion. While marked variability in IL-2 and IFN-γ expression was observed in virus-infected animals, notwithstanding the sample size, the increase in plasma levels of the two cytokines appear to be associated with a shorter viraemia. This may suggest that, in addition to virus neutralising antibodies, potential

IL-2 and IFN-γ-mediated cellular mechanisms may be required for the resolution of viraemia in BEF.

It is noteworthy to mention that the sample size used in the cytokine studies was unavoidably smaller than ideal. At the beginning of the research project, the initial plan was to conduct the cytokine studies on several experimentally infected cattle.

However, since the initial inoculation did not lead to clinical disease as per requirement of the project, a decision was made to study naturally infected cattle.

Consequently, two cohorts of sentinel cattle were studied during BEF outbreaks that occurred at two different time points. The two cohorts were made up of 24 and 18 animals respectively. Quite notably, only six animals from the first cohort and eight from the second cohort (total n=14) met the inclusion criteria. The other 28 animals were excluded after they tested positive for at least one of other non-BEFV regionally important arboviruses listed under materials and methods. Future studies based on a larger sample size are strongly recommended to further corroborate the cytokine study findings presented in this thesis.

5.2 Clarification of the tissue tropism and potential replication sites of the

BEFV

While a number of studies spanning several decades focused on describing the clinical and pathological aspects of BEF in cattle, (Basson et al., 1970; Burgess and

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Spradbrow, 1977; Mackerras et al., 1940; Young and Spradbrow, 1990), by the time of initiating the present research, major gaps still remained in the general understanding of the pathogenesis of the disease in cattle. Of particular interest, the potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of a BEFV infection have never been fully defined in cattle and therefore were not known. As reported in the literature, fluorescent antibody test has been previously used to demonstrate BEF viral antigen in tissues and within cellular sediments of serosal fluids taken from BEFV-infected cattle

(Burgess and Spradbrow, 1977; Theodoritis, 1969; Young and Spradbrow, 1985). In the study reported by Burgess and Spradbrow (1977), viral antigen appeared to be intracytoplasmically located within neutrophils as well as reticular cells in the spleen and lymph node. Other than these findings, however, tissue tropism and potential replication sites of the virus remained undefined until the present research was initiated. Therefore, one of the study objectives was designed to clarify the knowledge gaps on tissue tropism and potential in vivo tissue replication sites of

BEFV. To address this objective, biological specimens collected from naturally infected cattle were tested by a combination of laboratory techniques that included qRT-PCR, IHC, virus isolation, and electron microscopy. On the basis of the data obtained, BEFV appears to have preferential tropism for lymphoid tissues in cattle with the spleen and haemal node most likely to be potential in vivo replication sites for the virus. These conclusions are underpinned by the evidence of viral antigen in lymphoid tissues by IHC, by the isolation of BEFV RNA from several splenic and haemal node samples of post-viraemic cattle, as well as by the isolation of BEFV from an RNA-positive spleen of one animal whose haemal node was also positive for rhabdoviral particles six days after cessation of viraemia. On one hand, the present

122 data justify the potential use of lymphoid tissues as specimens of choice in post- mortem diagnostic investigations, especially where other biological samples are not available. Such diagnostic investigations should be based on various techniques including but not limited to BEFV-specific qRT-PCR, IHC, and virus isolation. Since the publication of the research data, two veterinary pathologists in Australia have reported positive qRT-PCR test results by testing spleens of post-viraemic cattle

(personal communication). On the other hand, the presence of viral antigen and RNA in the spleen and haemal node during the post-viraemic period of BEFV-infected cattle also throws more light as to the possible site(s) for the development of the immune response in this disease. Based on these findings, it is likely that BEFV may transiently localise and possibly replicate in lymphoid tissues beyond the viraemic phase of BEFV infections in cattleTherefore, post-mortem sampling of the spleen and haemal node are recommended, especially when other biological samples are not available to provide a reliable retrospective diagnosis in animals dying from suspected BEF. From a biosecurity perspective, however, it is highly unlikely that the presence of viral antigens and RNA in the meat may be of any concern, since

BEFV RNA is rapidly inactivated by the low pH developing in skeletal muscles during the post-slaughter period Additional studies are required to define the precise role of

T cells in anti-BEFV adaptive immune responses, as well as to confirm potential in vivo replication sites of BEFV in cattle by in situ hybridisation and other molecular tools.

5.3 Clarification on neuropathology and possible neurovirulence of BEFV

Neuromuscular weakness, paresis, and paralysis have been described as part of a wide spectrum of clinical signs seen in some BEFV-infectedcattle (St George et al.,

1986; St George et al., 1989; Tzipori, 1975). In a study reported by St George et al.

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(1986), hypocalcaemia was mentioned as a potential contributing factor to neuromuscular weakness and possibly paresis. However, such hypocalcaemia did not account for the paralysis that was seen in some BEFV-infected that were otherwise normocalcaemic (St George et al., 1986). As was shown in some of those studies, hypocalcaemia-induced muscle weakness and paresis were easily reversed by the parenteral infusion of calcium borogluconate (Nandi and Negi, 1999; St

George et al., 1989).

The first study to suggest that BEFV infection in cattle might be associated with encephalitis was reported by Tzipori (1975) who described fatal encephalitis experimentally induced by intracerebral inoculation of a virulent BEFV strain into seronegative calves but not in an age-matched negative controls. Subsequent to that report, severe bilaterally symmetrical Wallerian degeneration (WD) was also demonstrated in spinal cord sections of four BEFV-infected animals and these authors speculated that compressive myelopathy was the likely cause of the lesions and associated neurological symptoms (Hill and Schulz, 1977). Against this background, the studies conducted in this part of the thesis were carried out to assess the potential neurotropism of BEFV in naturally infected cattle. Additionally, the various histomorphological abnormalities detectable in the central and peripheral nerve tissues were described to see if a causal relationship could be established between these lesions and chronic paralysis in few of the BEFV-infected animals that become downers. The research data obtained confirm that BEFV is neurotropic in cattle and further suggest that, in addition to WD in the spinal cord, peripheral neuropathy and non-suppurative brain stem encephalitis may occur and could potentially contribute to chronic paralysis seen in BEFV-infected downer cattle.

124

These findings may explain paresis and paralysis observed in BEFV-infected-downer cattle that otherwise may be normocalcaemic. Since the present studies were based on case control studies, thesedata need to be further confirmed in experimental studies based on a much larger sample size. In summary, the research data presented in this thesis has bridged decades-old knowledge gaps in our understanding of the pathogenesis of BEF in cattle. New hypotheses have been generated for a better and comprehensive understading of the pathogenesis of this infectious disease in cattle.

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CHAPTER 6

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ANIMAL ETHICS APPROVAL No. 1

148

149

APPENDIX 2 – ANIMAL ETHICS APPROVAL No. 2

150

APPENDIX 3 – RAW qRT-PCR DATA

www.qiagen.com

Quantitation Report

Experiment Information

Run Name BEFV QDPI 2013-09-16 (1) Run Start 16/09/2013 2:57:34 PM Run Finish 16/09/2013 5:05:30 PM Operator Susan Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 9.33

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 5% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Messages

Message

Profile

Cycle Cycle Point Hold @ 50°c, 10 min 0 secs

151

Hold 2 @ 95°c, 10 min 0 secs Cycling (50 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 60°c, hold 60 secs, acquiring to Cycling A([Green][1][1]) Hold 3 @ 40°c, 0 min 20 secs

Raw Data For Cycling A.Green

Quantitation data for Cycling A.Green

No. Colour Name Type Ct RESULTS 1

152

No. Colour Name Type Ct RESULTS 4

5 2013/1023 - Gluteal nerve Unknown 23.92 DETECTED

6 2013/1023 - Liver Unknown 15.04 DETECTED

7 2013/1023 - Lung Unknown 18.52 DETECTED

8 2013/1023 - Kidney Unknown 21.51 DETECTED

9 2013/1023 - Synovial membrane Unknown 23.76 DETECTED

10 2013/1023 - Lumbar Spinal cord Unknown 25.24 DETECTED

11 2013/1023 - Thoracic Spinal cord Unknown 28.70 DETECTED

12 2013/1023 - Cervical Spinal cord Unknown 24.75 DETECTED

13 2013/1023 - Medulla Unknown 24.62 DETECTED

14 2013/1023 - Cerebellum Unknown 25.92 DETECTED

15 2013/1023 - Cerebrum Unknown 23.41 DETECTED

16 2013/1023 - Heart Unknown 24.21 DETECTED

17 2013/1023 - Skeletal muscle Unknown 24.78 DETECTED

18 2013/1023 - Tongue Unknown 26.00 DETECTED

19 2013/1023 - Pericardial fluid Unknown 28.39 DETECTED

20 2013/1023 - Synovial fluid Unknown 27.87 DETECTED

153

No. Colour Name Type Ct RESULTS

29 PBS Negative Control Not detected

30 BEF RNA Extraction control Positive Control 27.08 DETECTED

31 BEF RNA PCR control Positive Control 26.75 DETECTED

32 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

154

www.qiagen.com

Quantitation Report

Experiment Information

Run Name BEFV QDPI 2012-07-23 (1) Run Start 23/07/2012 12:03:53 PM Run Finish 23/07/2012 2:32:11 PM Operator Rachel Notes Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 8.

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction No No Template Control Threshold 10% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Profile

Cycle Cycle Point Hold @ 50°c, 30 min 0 secs Hold 2 @ 95°c, 10 min 0 secs Cycling (50 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 60°c, hold 60 secs, acquiring to Cycling A([Green][1][1]) Hold 3 @ 40°c, 0 min 20 secs

Raw Data For Cycling A.Green

155

Quantitation data for Cycling A.Green

No. Colour Name Type Ct Rep. Ct Result

1 2012/601 EDTA Unknown Not detected

2 PBS Negative Control Not detected

3 RNA 31.3.10 Positive Control 27.66 27.66 DETECTED

4 RNA PCR control #5 17.7.12 Positive Control 31.64 31.64 DETECTED

5 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

156

www.qiagen.com

Quantitation Report

Experiment Information

Run Name BEFV QDPI 2013-11-04 (1) Run Start 4/11/2013 4:04:38 PM Run Finish 4/11/2013 6:14:12 PM Operator Susan Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 8.

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 10% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Messages

Message

Profile

Cycle Cycle Point Hold @ 50°c, 10 min 0 secs Hold 2 @ 95°c, 10 min 0 secs

157

Cycling (50 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 60°c, hold 60 secs, acquiring to Cycling A([Green][1][1]) Hold 3 @ 40°c, 0 min 20 secs

Raw Data For Cycling A.Green

Quantitation data for Cycling A.Green

Standard Curve

158

No. Colour Name Type Ct RESULTS 1

2 2013/1213 - 50 - EDTA Unknown Not detected

3 2013/1213 - 50 - Pericardial Fluid Unknown Not detected

4 2013/1213 - 50 - Synovial Fluid Unknown Not detected

5 2013/1213 - 50 - Thoracic Fluid Unknown Not detected

6 2013/1213 - 50 - Peritoneal Fluid Unknown Not detected

7 2013/1213 - 50 - Prefemoral Lymph node Unknown Not detected

8 2013/1213 - 50 - Heart Unknown Not detected

9 2013/1213 - 50 - Skeletal muscle Unknown Not detected

10 2013/1213 - 50 - Kidney Unknown Not detected

11 2013/1213 - 50 - Spleen Unknown 33.11 DETECTED

12 2013/1213 - 50 - Cervical Spinal cord Unknown Not detected

13 2013/1213 - 50 - Medulla Unknown Not detected

14 2013/1213 - 50 - Gluteal nerve Unknown Not detected

15 2013/1213 - 50 - Haemonode Unknown 34.54 DETECTED

16 2013/1213 - 50 - Urinary Bladder Unknown Not detected

17 2013/1213 - 50 - Right Synovial Membrane Unknown Not detected

18 2013/1213 - 50 - Left Synovial Membrane Unknown Not detected

19 2013/1213 - 50 - Liver Unknown Not detected

20 2013/1213 - 50 - Lung Unknown Not detected

21 2013/1213 - 50 - Tongue Unknown Not detected

159

No. Colour Name Type Ct RESULTS

22 2013/1213 - 50 - Thoracic Spinal cord Unknown Not detected

23 2013/1213 - 50 - Lumbar Spinal cord Unknown Not detected

24 2013/1213 - 50 - Cerebrum Unknown Not detected

25 2013/1213 - 50 - Cerebellum Unknown Not detected

26 PBS Negative Control Not detected

30 2013/1236 - B103 - EDTA Unknown Not detected

31 2013/1236 - B103 - Pericardial Fluid Unknown Not detected

32 2013/1236 - B103 - Right Synovial Fluid Unknown Not detected

33 2013/1236 - B103 - Left Synovial Fluid Unknown Not detected

34 2013/1236 - B103 - Thoracic Fluid Unknown Not detected

35 2013/1236 - B103 - Peritoneal Fluid Unknown Not detected

36 2013/1236 - B103 - Prefemoral Lymph node Unknown Not detected

37 2013/1236 - B103 - Heart Unknown Not detected

38 2013/1236 - B103 - Skeletal muscle Unknown Not detected

39 2013/1236 - B103 - Kidney Unknown Not detected

40 2013/1236 - B103 - Spleen Unknown Not detected

41 2013/1236 - B103 - Cervical Spinal cord Unknown Not detected

42 2013/1236 - B103 - Medulla Unknown Not detected

43 2013/1236 - B103 - Gluteal nerve Unknown Not detected

44 2013/1236 - B103 - Haemonode Unknown Not detected

45 2013/1236 - B103 - Urinary Bladder Unknown Not detected

46 2013/1236 - B103 - Right Synovial Membrane Unknown Not detected

160

No. Colour Name Type Ct RESULTS

47 2013/1236 - B103 - Left Synovial Membrane Unknown Not detected

48 2013/1236 - B103 - Liver Unknown Not detected

49 2013/1236 - B103 - Lung Unknown Not detected

50 2013/1236 - B103 - Tongue Unknown Not detected

51 2013/1236 - B103 - Lumbar Spinal cord Unknown Not detected

52 2013/1236 - B103 - Cerebrum Unknown Not detected

53 2013/1236 - B103 - Cerebellum Unknown Not detected

54 PBS Negative Control Not detected

67 PBS Negative Control Not detected

70 BEF RNA Extracted Positive Control 30.31 DETECTED

71 BEF RNA PCR Positive Control 27.26 DETECTED

161

No. Colour Name Type Ct RESULTS

72 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

162

www.qiagen.com

Quantitation Report

Experiment Information

Run Name BEFV QDPI 2014-02-04 (1) Run Start 4/02/2014 2:20:26 PM Run Finish 4/02/2014 4:29:06 PM Operator Nikki Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 9.33

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 0% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Profile

Cycle Cycle Point Hold @ 50°c, 10 min 0 secs Hold 2 @ 95°c, 10 min 0 secs Cycling (50 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 60°c, hold 60 secs, acquiring to Cycling A([Green][1][1]) Hold 3 @ 40°c, 0 min 20 secs

Raw Data For Cycling A.Green

163

Quantitation data for Cycling A.Green

No. Colour Name Type Ct RESULTS

1 2014/0055 B121 Synovial fluid Unknown Not detected

2 2014/0055 B121 Pericardial fluid Unknown Not detected

3 2014/0055 B121 Thoracic fluid Unknown Not detected

4 2014/0055 B121 Peritoneal fluid Unknown Not detected

5 2014/0055 B121 EDTA blood Unknown Not detected

6 2014/0055 B121 Cerebellum Unknown Not detected

7 2014/0055 B121 Cerebrum Unknown Not detected

164

No. Colour Name Type Ct RESULTS

8 2014/0055 B121 Spinal cord cervical Unknown Not detected

9 2014/0055 B121 Nerve gluteal Unknown Not detected

10 2014/0055 B121 Haemonode Unknown 31.46 DETECTED

11 2014/0055 B121 Heart Unknown Not detected

12 2014/0055 B121 Kidney Unknown Not detected

13 2014/0055 B121 Liver Unknown Not detected

14 2014/0055 B121 Spinal cord lumber Unknown Not detected

15 2014/0055 B121 Lung Unknown Not detected

16 2014/0055 B121 Medulla Unknown Not detected

17 2014/0055 B121 Lymph node prefemoral Unknown Not detected

18 2014/0055 B121 Muscle skeletal Unknown Not detected

19 2014/0055 B121 Spleen Unknown Not detected

20 2014/0055 B121 Synovial membrane left Unknown Not detected

21 2014/0055 B121 Synovial membrane right Unknown Not detected

22 2014/0055 B121 Spinal cord Unknown Not detected

23 2014/0055 B121 Tongue Unknown Not detected

24 2014/0055 B121 Bladder Unknown Not detected

25 2014/0055 B121 Synovial fluid left stifle Unknown Not detected

26 PBS Negative Control Not detected

165

No. Colour Name Type Ct RESULTS 33

34 PBS Negative Control Not detected

35 BEF RNA Extraction control Positive Control 27.97 DETECTED

36 BEF RNA PCR control Positive Control 26.60 DETECTED

37 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

166

www.qiagen.com

Quantitation Report

Experiment Information

Run Name BEFV QDPI 2014-03-12 (1) Run Start 12/03/2014 2:24:59 PM Run Finish 12/03/2014 4:33:38 PM Operator Susan Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 8.

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 0% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Messages

Message

Profile

167

Raw Data For Cycling A.Green

Quantitation data for Cycling A.Green

No. Colour Name Type Ct RESULTS

1 2014/0166 - EDTA Unknown Not detected

2 2014/0166 - L stifle joint fluid Unknown Not detected

3 2014/0166 - R stifle joint fluid Unknown Not detected

4 2014/0166 - Pericardial Fluid Unknown Not detected

5 2014/0166 - Thoracic Fluid Unknown Not detected

6 2014/0166 - Peritoneal Fluid Unknown Not detected

168

No. Colour Name Type Ct RESULTS

7 2014/0166 - Liver Unknown Not detected

8 2014/0166 - Lung Unknown Not detected

9 2014/0166 - Kidney Unknown Not detected

10 2014/0166 - Bladder Unknown Not detected

11 2014/0166 - Spleen Unknown Not detected

12 2014/0166 - Haemonode Unknown Not detected

13 2014/0166 - Heart Unknown Not detected

14 2014/0166 - Skeletal muscle Unknown Not detected

15 2014/0166 - Tongue Unknown Not detected

16 2014/0166 - L Stifle joint Unknown Not detected

17 2014/0166 - R Stifle joint Unknown Not detected

18 2014/0166 - Prefemoral Lymph node Unknown Not detected

19 2014/0166 - Gluteal nerve Unknown Not detected

20 2014/0166 - Cervical spinal cord Unknown Not detected

21 2014/0166 - Thoracic spinal cord Unknown Not detected

22 2014/0166 - Lumber spinal cord Unknown Not detected

23 2014/0166 - Cerebrum Unknown Not detected

24 2014/0166 - Cerebellum Unknown Not detected

25 2014/0166 - Medulla Unknown Not detected

27 PBGS Negative Control Not detected

28 BEF RNA 15.10.13 Extracted Positive Control 27.71 DETECTED

29 BEF RNA 15.10.13 PCR Positive Control 26.94 DETECTED

30 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

169

170

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Quantitation Report

Actions for reporting Date Initial Positive Control within limits & entered onto MU (x3) Results entered onto LADS

Experiment Information

Run Name BEFV QDPI 2014-12-02 (1) Run Start 2/12/2014 4:18:25 PM Run Finish 2/12/2014 4:30:33 PM Operator Susan Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 8.

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 10% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Messages

Message

Profile

Cycle Cycle Point Hold @ 50°c, 10 min 0 secs Hold 2 @ 95°c, 10 min 0 secs

171

Cycling (50 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 60°c, hold 60 secs, acquiring to Cycling A([Green][1][1]) Hold 3 @ 40°c, 0 min 20 secs

Raw Data For Cycling A.Green

Quantitation data for Cycling A.Green

No. Colour Name Type Ct RESULT 1

172

No. Colour Name Type Ct RESULT 5

6 2014/1070 - 577 - EDTA Unknown Not detected

7 2014/1070 - 577 - Serum Unknown Not detected

8 2014/1070 - 577 - Synovial fluid - left stifle joint Unknown 32.05 DETECTED

9 2014/1070 - 577 - Synovial fluid - right stifle joint Unknown 36.85 DETECTED

10 2014/1070 - 577 - Unknown Not detected

11 2014/1070 - 577 - Unknown Not detected

12 2014/1070 - 577 - Unknown Not detected

13 2014/1070 - 577 - Unknown Not detected

14 2014/1070 - 577 - Unknown Not detected

15 2014/1070 - 577 - Unknown Not detected

16 2014/1070 - 577 - Unknown Not detected

17 2014/1070 - 577 - Unknown Not detected

18 2014/1070 - 577 - Unknown Not detected

19 2014/1070 - 577 - Unknown Not detected

20 2014/1070 - 577 - Spleen Unknown 36.04 DETECTED

21 2014/1070 - 577 - Haemal node Unknown 32.57 DETECTED

22 2014/1070 - 577 - Unknown Not detected

23 2014/1070 - 577 - Unknown Not detected

24 2014/1070 - 577 - Unknown Not detected

25 2014/1070 - 577 - Unknown Not detected

26 2014/1070 - 577 - Unknown Not detected

27 2014/1070 - 577 - Unknown Not detected

28 2014/1070 - 577 - Unknown Not detected

29 2014/1070 - 577 - Unknown Not detected

173

No. Colour Name Type Ct RESULT

30 2014/1070 - 577 - Unknown Not detected

31 2014/1070 - 577 - Unknown Not detected

32 2014/1070 - 577 - Unknown Not detected

33 2014/1070 - 577 - Unknown Not detected

34 2014/1070 - 577 - Unknown Not detected

35 2014/1070 - 577 - Unknown Not detected

36 2014/1070 - 577 - Unknown Not detected

37 2014/1070 - 577 - Unknown Not detected

38 2014/1070 - 577 - Unknown Not detected

39 2014/1070 - 577 - Unknown Not detected

40 2014/1070 - 577 - Unknown Not detected

41 2014/1070 - 577 - Unknown Not detected

42 2014/1070 - 577 - Unknown Not detected

43 2014/1070 - 577 - Unknown Not detected

44 2014/1070 - 577 - Prefemoral lymph node - left Unknown 33.51 DETECTED

45 2014/1070 - 577 - Prefemoral lymph node - right Unknown 33.76 DETECTED

46 2014/1070 - 577 - Unknown Not detected

47 2014/1070 - 577 - Unknown Not detected

48 2014/1070 - 577 - Unknown Not detected

49 2014/1070 - 577 - Unknown Not detected

50 2014/1070 - 577 - Unknown Not detected

51 2014/1070 - 577 - Unknown Not detected

52 2014/1070 - 577 - Unknown Not detected

53 2014/1070 - 577 - Unknown Not detected

54 2014/1070 - 577 - Unknown Not detected

174

No. Colour Name Type Ct RESULT

55 2014/1070 - 577 - Unknown Not detected

56 2014/1070 - 577 - Unknown Not detected

57 2014/1070 - 577 - Unknown Not detected

58 2014/1070 - 577 - Unknown Not detected

59 2014/1070 - 577 - Unknown Not detected

60 2014/1070 - 577 - Unknown Not detected

61 2014/1070 - 577 - Unknown Not detected

62 PBGS Negative Control Not detected

63 PBS Negative Control Not detected

64 RNA 15.10.13 Extracted Positive Control 27.92 DETECTED

65 RNA 15.10.13 Extracted Positive Control 28.11 DETECTED

66 RNA 15.10.13 PCR Positive Control 27.05 DETECTED

67 NTC NTC Not detected

68 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

175

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Quantitation Report

Experiment Information

Run Name BEFV QDPI 2013-05-10 (1) Run Start 10/05/2013 12:21:29 PM Run Finish 10/05/2013 2:50:36 PM Operator Nikki Notes BEFV QDPI Run On Software Version Rotor-Gene 2.0.3.2 Run Signature The Run Signature is valid. Gain Green 10.

Quantitation Information

Threshold 0.050 Left Threshold 1.000 Standard Curve Imported No Standard Curve (1) N/A Standard Curve (2) N/A Start normalising from cycle 1 Noise Slope Correction Yes No Template Control Threshold 10% Reaction Efficiency Threshold Disabled Normalisation Method Dynamic Tube Normalisation Digital Filter Light Sample Page Page 1 Imported Analysis Settings

Profile

176

Raw Data For Cycling A.Green

Quantitation data for Cycling A.Green

No. Colour Name Type Ct Results 1

3 13/546 EDTA Blood Neat Unknown Not Detected

4 13/546 Pericardial fluid Neat Unknown Not Detected

5 13/546 Thoracic fluid Neat Unknown Not Detected

6 13/546 Synovial fluid (Hip) Neat Unknown Not Detected

177

No. Colour Name Type Ct Results

7 13/546 Synovial fluid (stifle) Neat Unknown Not Detected

8 13/546 Peritoneal fluid Neat Unknown Not Detected

9 13/546 Amniotic fluid Neat Unknown Not Detected

10 13/546 EDTA Blood Unknown Not Detected

11 13/546 Pericardial fluid Unknown Not Detected

12 13/546 Thoracic fluid Unknown Not Detected

13 13/546 Synovial fluid (Hip) Unknown Not Detected

14 13/546 Synovial fluid (stifle) Unknown Not Detected

15 13/546 Peritoneal fluid Unknown Not Detected

16 13/546 Amniotic fluid Unknown Not Detected

23 PBS Negative Control Not Detected

24 BEF RNA Ext Control 19/11/12 Positive Control 27.05 Detected

25 BEF RNA PCR Control Positive Control 26.71 Detected

26 NTC NTC Not Detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

178

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Quantitation Report

Experiment Information

Quantitation Information

Profile

Raw Data For Cycling A.Green

179

Quantitation data for Cycling A.Green

No. Colour Name Type Ct Rep. Ct Result

1 2012/601 EDTA Unknown Not detected

2 PBS Negative Control Not detected

3 RNA 31.3.10 Positive Control 27.66 27.66 DETECTED

4 RNA PCR control #5 17.7.12 Positive Control 31.64 31.64 DETECTED

5 NTC NTC Not detected

Legend: NEG (NTC) - Sample cancelled due to NTC Threshold. NEG (R. Eff) - Sample cancelled as efficiency less than reaction efficiency threshold.

180

181