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EPIDEMIOLOGY OF EQUINE HERPESVIRUS INFECTIONS 
ZHEN FANG FU 
[jb; 
A thesis presented in partial fulfilment (75%) 
of the requirement for the degree of 
Master of Philosophy in Veterinary 
Science at Massey University 
January 1 985 
ABSTRACT 
The epi demi ol ogy of i nfecti ons wi th e qu i ne herpesvi rus ( EHV ) 
t ypes 1 and 2 i n  foals on a Thoroughbred stud i n  New Zeal and  was 
i nvesti gated . As part of this  study  an ELISA test was devel oped to 
measure anti body ti tr es to  EHV- 2 in e qui ne  sera . All the sera 
coll ected f rom the f oals befor e the i ngesti on of col ostrum wer e 
negati ve for anti bodi es to both EHV-1 and EHV-2 .  S oon aft er 
sucki ng ,  these foals had serum ant i body l evels agai ns t  these two 
vi ruses simil ar to  those of the i r  dams . The maternal ly  deri ved 
ant i body to EHV- 1  l asted for 3-4 months and anti body t i tres rose 
a gai n at around weani ng time . In contr ast , passi vel y ac qui red 
anti body to EHV-2 was rapi dly  supplemented by act i vely  produced 
anti body . 
Serol ogi cal evi dence suggested that most  of the f oals ( 85%) 
became  i nfected  wi th EHV-1 ,  and 25%  wer e rei nfected i n  the i r  f i rst 
t en months of l if e ; however EHV- 1 was not recovered e i ther f rom 
these mar es or thei r foals duri ng the i nvest i gation peri od despi te  
t he l arge i ncrease in  anti body t i tres .  Ser ol ogi cal evi dence of 
EHV-1 i nf ecti on i n  foals i ndi cat ed  that this occurred  around the 
time of weani ng when the maternal l y  deri ve d  antibody had decl i ned t o  
a level whi ch was pr esumabl y unprote cti ve . The cl i ni cal si gns whi ch 
devel oped after EHV- 1 i nfect i on wer e  very  m i l d ,  the ma in  symptom 
observed bei ng a profuse nasal discharge usuall y  l asti ng two or 
three days , occasi onally  wi th an el evati on of body t emper atur e .  The 
source of EHV- 1 i nfection  i n  foals could not be determined and there  
was no evi dence to  suggest that the i r  dams were i nf ected wi th EHV- 1 
around the time when the f oals becam e  i nfected . However , a 
3 
rel ationship between prei nfection  anti body t i tr es ( l og 1 0  ) agai nst 
EHV- 1 and the vi ral i nf ecti on was observe'd . 
I n  cont r ast , EHV- 2 was isol ated from all of the foals by 2 to 4 
months of age . The v i rus i nf ecti on persisted i n  these ani mals f or 2 
to 6 months and stimul ated conti nuous production of anti body . As 
soon as the ant i body l evel agai nst EHV-2 reached a peak , the 
i i 
i i i 
i sol ation of the virus decreased , an d eventual ly EHV- 2 was no l onger 
i sol ated f rom these f oal s by 9 months of age . The f oal s possi bl y  
dams  
EHV-2 
contracted EHV- 2 i nfection from their 
e xcr eted the vi rus ar ound the t ime  when 
thei r foals . Cl i ni cal reacti ons at  
i nfect i on var i ed f rom f oal to  f oal,  
around 
rangi ng 
since some  of them 
was i sol at ed f rom 
the time  of  EHV- 2 
f rom subcl i ni cal to 
fever , mucopurul ent nasal di schar ge ar.d swell en submandi bular l ymph 
nodes . Two s everely  aff ected f oal s f rom whi ch EHV-2 was i sol at ed 
d ied  of compl ications r esul ti ng from secondary bacteraemi a .  F rom 
t hes e f i ndi ngs , an associ at i on between EHV-2 and the r es pi rat ory 
di sease obs erved i n  these foals  was postulated . H owever , the 
possi bl e  r ol e  of EHV-2 as a pathogen f or young f oal s needs 
conf i rmati on by f urther studi es i ncl udi ng e xperimental i nfection of 
gn,otob iot i c  f oal s .  
A trai l for eval uat i on of P neumabort- K (an EHV- 1 s ubtype 
vacci ne ) was conducted  i n  these f oal s .  A nimal s  inoculated  wi th the 
vacci ne at the age of 30 and 60 days f ai l ed t o  respond s er ol ogically  
to  the immuni zation , and i t  was assume d that thi s  was d ue to  the 
i nteref erence of the high l evel s  of pass i vely acqu i red ant i body . 
Based on thi s observation ,  another EHV- 1 vaccin ati on procedure  f or 
f oal s commenci ng at 80 -90 days was recommende d.  
iv 
ACKNOWLEDGEMENT 
The work described in this thesis was jointly supported by a 
China-NZ exchange scholarship , the Department of· Vete rinary Patholgy 
and Public Health, Massey University , and Mr . D . Benjamin of Field 
House , Matamata . 
Thanks are due to Professor B .W .Manketelow who. kindly made  
available the facilities of the Department of  Veterinary Pathology 
and Public Health, Massey Unive rsity for this study . 
I would particularly like to thank Dr . R . B . Marshall ,  my 
supervisor ,  for his continuous help and encouragement throughout 
this project ; Dr . D . J . Hampson for his commentary on some of the 
experiments , and particularly in the preparation of  this thesis; 
Dr . M . R . Al ley for his help whenever  needed ; and Mrs . Linley Denby 
for her excellent technical assistence . 
The friendship and effort o f  the staff of  
Veterinary Services , involving collec tion of all  
useful d iscussions and generous hospitality , 
appreciated , and special thanks are due to Merss 
J . B . Grimmett , and P . D . Jolly.  
the Ma tama ta 
the mate rials ,  
are greatly 
L . G . Dickinson , 
I am also indeted to Dr . A . J . Robinson , director o f  MRC Virus 
Research Uni t ,  for his enthusiasm and useful suggestions ; Mr . 
G .W . Horner ,  Veterinary Investigation Officer o f  Ruakura Animal 
Health Laboratory , Hamilton for advice and kindly suppling EHV- 1 and 
EHV-2 viruses .  
Finally , I would also like to thank all the staff in the 
Department of  Veterinary Pathology and Public Health,  especially 
Professor D .K . Blackmore for guidance in Epidemiology , Dr . 
K .M .Moriaty for advice in Immunology , Dr . G.V. Peterson for help 
with statistical analysis , Mr. P . N .Wildbore for administrative 
assistance ,  and Mr . T . Law for the preparation of the pho tographs . 
Chapter 1 
Chapter 2 
2 .  1 
2 .  1 .  1 
2 .  1 .  2 
2 . 1 . 3 
2 .  1 .  4 
2 . 2  
2 . 2 . 1 
TABLE OF CONTENTS 
ABSTRACT 
ACKNOWLEDGEMENTS 
TABLE OF CONTENTS 
INDEX OF TABLES 
INDEX OF FIGURES AND PLATES 
INTRODUCTION 
LITERATURE REVIEW 
Equine Herpesviruses 
Introduction 
Virion Mophology and Structure 
Resistance of EHVs to Inactivation 
Propagation of EHV 
Equine Herpesvirus Type 
Clinical Manifestations and Pathology of EHV- 1 
Infection 
Respiratory Disease 
Abortion 
Perinatal Mortality 
Paralysis 
2 . 2 . 2  Epidemiology of EHV- 1 Infection 
2 . 2 . 3  ·Immunity to EHV- 1 Infection 
2 . 2 . 4  Immunization against EHV- 1 Infection 
2 . 3  Equine Herpesvirus Type 2 
2 . 3 . 1 Introduction 
2 . 3 . 2  Clinical signs and pathology 
2 . 3 . 3 Epidemiology and Immunology of EHV-2 Infection 
V 
i i  
iv 
V 
ix 
X 
1 
4 
4 
4 
4 
6 
1 
1 0  
1 0  
1 0  
1 2  
1 3  
1 5  
1 6  
2 0  
2 3  
27 
27 
27  
3 1  
Chapter 3 MATERIALS and METHODS · 
3 . 1 Animals  
vi  
35  
35  
3 . 2  Sampling Procedures for Horses 35 
3 . 2 . 1  Nasal swabs 35 
3 . 2 . 2  Sera 36 
3 . 3  Cell Culture 36 
3 . 3 . 1 Storing and Reconstitution of cells 36 
3 . 3 . 2  Growth and Trypsinization of Confluent Monolayers 37 
3 . 4  Virus Isolation 37 
3 . 5 Production of Virus Stocks 38 
3 . 5 . 1 EHV- 1 38 
3 . 5 . 2  EHV-2 38 
3 . 6  Titration of Viruses 39  
3 . 7  Virus Identification 39 
3 . 7 . 1 Observation of  Virus Behaviour in Cell Culture 39 
3 . 7 . 2  Electron Microscopy (EM) 39 
3 . 7 . 3  Chloroform Sensitivity 40 
3 . 7 . 4  Staining of Inclusion Bodies 40 
Chapter 4 THE DEVELOPEMENT OF AN ELISA TEST FOR DETECTING 
ANTIBODIES AGAINST EHV-2 IN EQUINE SERA 
4 . 1 Introduction 
4 . 2  Materials and Methods 
4 . 2 . 1  _Preparation of Antigen 
4 . 2 . 2  ELISA Procedures 
4 . 2 . 3  Standardization of  ELISA 
.A Titration of Serum and Conjugate 
B Determination of  Optimum Antigen Concentration 
C Colour Development 
4 . 2 . 4 Titration of Test  Sera 
4 . 2 . 5  Expression of the ELISA Result 
4 . 2 . 6  Comparision of  ELISA Results with SN Results 
and Virus Isolation 
4 . 3  Results 
4 . 3 . 1  Antigen 
4 . 3 . 2 Standardization of  Serum and Conjugate 
4 . 3 . 3  Determination of  Optimum Antigen Concentration 
4 1  
4 1  
4 1  
4 1  
42 
43 
43 
44  
44 
45  
45  
46  
46 
46  
46  
•47 
4 . 3 . 4  
4 . 3 . 5  
4 . 3 . 6  
4 . 4  
Chapter 5 
5 .  1 
5 . 2  
5 . 3  
A 
B 
5 . 3 . 3 
A 
B 
c 
Colour Developement 
Titration of  Test Sera 
Coroparision of ELISA Titres with SN Titres 
Discussion 
EQUINE HERPESVIRUS TYPE 1 
Introduction 
Meterials and Methods 
Results 
Passive Immunity 
Response to Vaccination in Thoroughbreds 
Response of Mares to Vaccine 
Response of Foals to Vaccine 
Evidence of Infection with EHV- 1 
EHV- 1 Infection in Foals 
EHV- 1 Infection in  Mares 
Effect of Maternally  Derived Ant ibody on Response to 
Vaccination and the Occurrence of  EHV- 1 Infection in 
Thoroughbred Foals 
5 . 3 . 4  Rel�tionship between EHV-1 Infection and Respiratory 
5 . 4  
Chapter 6 
6 . 1 
6 . 2  
6 . 2 .  1 
6 . 2 . 2  
6 . 2 . 3  
A 
B 
6 . 2 . 4 
6 . 2 . 5  
6 . 3  
Disease 
Discussion 
EQUINE HERPESVIRUS 2 
Introduction 
Resul ts 
Maternally Derived Antibody 
Virus Isolation 
Serological Response 
Serological Response of Foals to EHV-2 Infection 
Serological Response of Mares to EHV-2 Infection 
Clinical Observation Associated with EHV-2 Infection 
in The Foals 
Characterization o f  Viral Isolates  
Discussion 
vii  
47  
48  
48  
49  
6 1  
6 1  
62 
63 
63  
63 
63  
63  
6 4  
64  
65  
65  
66  
66  
74  
74 
7 5  
7 5  
7 5  
7 6  
7 6  
7 6  
7 7  
7 8  
7 8  
Chapter 7 GENERAL CONCLUSION 
APPENDIX I 
APPENDIX I I  
REFERENCES 
viii 
94 
97 
1 0 0  
1 04 
Table 4- 1 
Table 4-2 
Table 4-3 
INDEX OF  TABLES 
Protein contents of viral and control antigens 
Serum-conjugate chequerboard titration 
Antibody titres to EHV-2 as measured by serum 
neutralisation ( SN)  test and by ELISA test  
Table 5- 1 Serum antibody titres against EHV- 1 in mares and 
in their foasls  after consumption of colostrum 
Table 5-2 Serum antibody titres against EHV- 1 in mares and 
in their foals at one month of age 
Table 6- 1 Serum antibody titres against EHV-2 in mares and 
in their foals after ingestion of colostrum 
Table 6-2 Recovery of EHV-2 from foals 
Table 6-3 Recovery of EHV-2 from mares 
ix 
52 
53  
54 
7 1  
72 
84 
85  
86 
X 
INDEX OF FIGURES AND PLATES 
Figure 4- 1 Comparison of ELISA activities at different dilution 
of serum and conjugate 55 
Figure 4-2 Comparison of ELISA activities at different antigen 
concentrations (strongly positive serum ) 56  
Figure 4-3 Comparison of ELISA activities at different antigen 
concentrations (weakly positive and negative sera )  57  
Figure 4-4  Titration curves of representative sera 58 
Figure 4-5 Comparison of SN and ELISA antibody titres against 
EHV-2 
Figure 4-6 Frequency distribution of  EHV-2 antibody titres 
Figure 5- 1 
Figure 6- 1 
Figure 6-2 
Figure 6-3 
obtained by SN and ELISA 
ELISA antibody titres agains t EHV- 1 in mares and 
their foals 
Incidence of  EHV-2 infection in foals 
Prevalence of EHV-2 infection in foals 
ELISA antibody titres against EHV-2 in mares and 
their foals 
59 
60 
73 
87 
88 
89 
Figure 
Figure 
6-4 
6-5 
Serological response of  foal No . 1 to EHV-2 infection 90 
Serological response of foal No . 1 1  to EHV-2 
infection 9 1  
Plate 6- 1 Characteristic CPE of EHV-2 ( 3  days post infection ) 92 
Plate 6-2 Characteristic CPE of EHV-2 (5 days post infection ) 92 
Plate 6-3 Cowdry type A inclusion bodies in EHV-2 infected 
cells 93 
Plate 6-4 Morphology of EHV-2 under EM (enveloped , empty 
particle ) 93