Applied veterinary Virlgy: The islatin and identificatin f viruses using embrynated Authrs: Prf. Estelle H Venter Licensed under a Creative Cmmns Attributin license. INTRODUCTION Viruses can nly grw in living cells but they are particular abut the type f cell they infect and grw in there is n universal cell that will supprt all viruses. Viruses tend t be hstspecific; therefre human viruses grw best in cells f human rigin, bvine viruses in bvine cells, canine viruses in canine cells, while sme viruses will nt grw in vitr at all. Therefre in the labratry the suspected virus must be grwn in a culture methd knwn t supprt its grwth. animals are used fr studying viruses which d nt grw in cell cultures r eggs, and fr testing vaccines, eggs supprt a fairly wide range f animal and human viruses hence their imprtance in the diagnstic service, cell cultures; different types f cell lines will supprt different types f viruses. The grwth f viruses in eggs Embrynated hen s eggs will supprt the grwth f sme viruses. Nt all viruses will grw in the tissues f embrynated eggs initially but many can be adapted t grwth in eggs withut much difficulty. Eggs prvide a suitable means fr the primary islatin and identificatin f viruses, the maintenance f stck cultures and the prductin f vaccines. The viruses grw in the cells f the embry and membranes and can be detected in several ways. These include mrtality, defrmity r haemrrhages in the embry, lesins n the membrane in the frm f pcks, edema f the develping membranes, inclusin bdies in sectins prepared frm embry tissues r the presence f viral antigens in the egg fluids. An embrynated hen s egg cntains cells (the embry and its membranes) that will supprt the grwth f sme viruses. They grw either in the cells f the embry r its membranes, r in bth and when these cells die they are liberated int the egg fluids. Cllectin f the virusinfected egg fluid is relatively simple, if smewhat messy. 1 P a g e
Eggs are inexpensive and easy t maintain. Eggs cme in a usually sterile package surrunded by a prus shell. As they shuld arrive clean in the labratry, they shuld nt be washed r immersed in water as this may allw bacteria t enter the egg. Use a quickly evaprating agent, such as alchl, tincture f idine r ether, t sterilize the eggshell at the site re inculatin. Eggs are freely available, especially hens eggs but ducks eggs have als been used. The immune system f the embry has nt matured; therefre antibdies are nt prduced against the inculated virus. Hwever, maternal antibdies are transferred frm the hen t the egg which implies that eggs shuld be btained frm nn-vaccinated (especially against Newcastle disease virus), mycplasma-free flcks. Advantages f eggs ver animal hst systems Eggs are readily available, cheap and easy t maintain Preliminary incubatin f the eggs is carried ut at 38 39 C and 60 70% humidity. The eggs need t be turned at least twice a day r rlled cntinually in a specially designed egg incubatr Once inculated, the eggs are incubated at temperatures suitable fr the grwth f the virus, but still maintaining a high degree f humidity Eggs are easily manipulated under sterile cnditins Eggs cme in a sterile package surrunded by a prus shell. They shuld arrive clean in the labratry. D nt wash the eggs r immerse them in water as this may allw bacteria t enter the prus shell. T sterilize the site f inculatin, use a quickly evaprating agent, such as alchl, tincture f idine r ether Eggs are sheltered frm the natural diseases ften bserved in labratry animals, and are relatively free frm bacterial and many latent viral infectins. Hwever maternal antibdies are transferred frm the mther hen, therefre the eggs shuld be btained frm nn-vaccinated (especially Newcastle disease virus), mycplasma-free flcks Eggs are generally free frm natural factrs f defence, specific r nn-specific, that smetimes intervenes and prevents passage in adult animals. The immune system f the embry has nt matured therefre antibdies are nt made against the inculated virus. Als the embry is sensitive f sme viruses that are harmless t the adult bird Eggs are easily identified and labelled with details f date, virus inculated and experimental prcedure Different rutes f inculatin fr different viruses are available i.e. 2 P a g e
the amnitic and / r allantic cavities, the ylk sac, the chri-allantic membrane, intravenus. Viruses that can grw in embrynated Embrynated are nt rutinely used fr the islatin f viruses. Viruses f veterinary imprtance which grw in eggs are tabulated under disease caused, age and rute f infectin, incubatin temperature and utcme f infectin. Table 1: Viruses f veterinary imprtance that can be grwn in embrynated ANIMAL VIRUS GENUS DISEASE ROUTE OF INOCULATION OUTCOME OF INFECTION Rhabdvirus Vesicular Stmatitis Inculate nt allantic Sac Embry death Cattle 8 ld embry 3 4 incubatin Capripxvirus Lumpy skin disease Inculate nt CAM 7 9 incubatin 33,5 35 C Orthpxvirus Cwpx Inculate nt CAM Can be distinguished frm pseudcwpx (parapx) which des nt grw n the chri-allantic membrane. Pcks / lesins n membrane Staining f membrane Pcks / lesins n membrane Swine Orthmyxvirus Swine influenza Amnitic/allantic inculatin int 9-10 day ld embrynating eggs Incubate 48 hrs at 33.5 C chick RBC Iridvirus African swine fever Ylk sac inculatin Causes death in 6 7 Sheep and gats Orbivirus Bluetngue Ylk sac / IV Flavivirus Wesselsbrn disease Ylk sac 10 11 ld embrynating eggs Incubate 3-5 at 33.5 C embry. Further prcessed fr presence f virus 3 P a g e
Orthmyxvirus Equine influenza Amnitic/Allantic inculatin int 10 day ld embrynating eggs chick and pig RBC. Hrses Incubate 48 hrs at 33 C Reviridae Orbivirus African hrse sickness Ylk sac /IV 10 12 ld embrynating eggs Incubate at 33 C fr 3 7 embry. Brnavirus CAM Dgs Paramyxvirus Canine distemper CAM needs adaptatin Prduces lesins Rhabdvirus: Lyssavirus Rabies Difficult, but can be adapted Paramyxvirus Newcastle disease Amnitic/allantic inculatin int 9-10 day ld embrynating eggs Incubate 2 5 at 37 C embry. chick RBC. Herpesvirus Infectius laryngtracheitis CAM plaque frmatin and death f embry Amnitic/allantic inculatin. Incubate 5-7 Crnavirus Infectius brnchitis Amnitic/allantic inculatin Incubate 30 hrs at 37 C Pultry Effects n embry include death, dwarfing, and curling, plus uratic depsits in the mesnephrns. Crnavirus Turkey enteritis (Bluecmb disease) Amnitic inculatin grws in embry intestines r ylk sac, 3 5 incubatin. Sme strains require 10 incubate fr maximum virus. Further prcessed fr presence f virus Orthmyxvirus Avian influenza, Myna, turkey, chicken, duck, gull. Amnitic/allantic inculatin Incubate 2 at 35 C Death f embry within 48 hurs. Adenvirus type 1 Chick embry lethal rphan (CELO) Avian adenvirus infectin Allantic inculatin Incubate 3-4 May need up t five blind passages embry with necrtic fci in liver and urate accumulatins in mesnephrns 4 P a g e
Px: Avipx Fwlpx CAM Prduces fcal r diffuse pcks Entervirus Avian encephalmyelitis Ylk sac inculatin Incubate 9 at 37 C Muscular dystrphy and paralysis. Signs f encephalmyelitis bserved after hatching. Entervirus Duck viral hepatitis Turkey hepatitis Allantic inculatin int 9-11day eggs the embry Revirus Infectius bursal disease CAM Miscellaneus Orthpx Ectrmelia (musepx) CAM Lepripx Myxmatsis (rabbits) CAM Lepripx Shpe fibrma N lesins Table 2: The use f eggs t distinguish between clinically similar diseases VIRUS AND DISEASE EFFECT ON: CHORIO- ALLANTOIC EFFECT ON: CHICKEN EMBRYO HAEMAGGLUTINATIO N OF CHICK EMBRYO FLUIDS Newcastle disease virus (NDV), a paramyxvirus f chickens Laryngtracheitis, a herpesvirus f chickens and pheasants Infectius brnchitis, a crnavirus f chickens N pcks Lethal within 48-72 hurs HA psitive Pcks N effect HA negative N pcks Dwarfing HA negative 5 P a g e