The occurrence of chronic and acute bee paralysis viruses in bees outside Britain

JOURR’AL

OF

INVERTEBRATE

The

PATHOLOGY

Occurrence Viruses

7, 167-169

of

(1965)

Chronic in

and

Bees

Acute

outside

Bee

Paralysis

Britain

L. BAILEY Rothamsted

Experimental .Accepted

Station, ,%‘ovember

Harpenden, 23,

England

1961

Honey bees (Apis mellifera Linnaeus) from Austria and Switzerland, suffering from and from Italy and Norway suffering from Ma1 Noir contained as much chronic bee paralysis virus as bees suffering from “paralysis” in Britain and Malta. These diseases appear to be etiologically the same, therefore, and the variable and unreliable signs sometimes exhibited are perhaps caused by factors secondary to infection by the virus. Apparently healthy bees from Canada and Italy were infected with the Grus of acute paralysis, as they are in Britain. Waldtrachtkrankheit,

INTRODUCTION

by injecting extracts of a sample of beesinto Chronic bee paralysis virus (CBPV) has live individuals of the samesample. This paper gives the results of tests for been identified as the cause of so-called CBPV in beesthat had diseasesdiagnosedin “paralysis” of honey bees (Apis nzellifera Europe or Norway as Waldtrachtkrankheit Linnaeus) in Britain and has been identified or Ma1 ~Voir before they were sent to me, and in one sample of diseasedhoney bees from of tests for ABPV in apparently healthy bees Hong Kong (Bailey et al., 1963; Bailey, sent from Canada and Italy. 1965). It has been considered a likely cause of the bee diseasesin Europe known as WaldMATERIAL AND METHODS trachtkrankheit and Ma1 Noir becauseMorison’s cell-inclusions (Morison, 1936) , which Two samples, from different colonies, of occur in bees with these diseases (Lotmar, bees suffering from Waldtrachtkrankheit 1940: Vecchi and Zambonelli, 1961), seem were received from Austria, one in June and one in July (1964) and another came from specifically associated with CBPV (Bailey, Switzerland in July. Two samples,from dif1965). Acute bee paralysis virus (ABPV) occurs ferent localities, of bees suffering from Mal commonly in honey bees and bumble beesin Noir were received from Italy, one in June Britain (Bailey and Gibbs, 1964). It has not and one in August. One sample of beeswith been associated with any disease seen in paralysis was obtained from a source in nature but it readily causes acute paralysis Britain in June and another came from Malta when about lo’ or more particles are injected in November. One sample of bees suffering from “Black Disease” was received from into the hemolymph of adult bees and the virus in apparently healthy bees can some- Norway in June. All these sampleswere tested times be caused to multiply and cause disease for CBPV. Ten bees of each sample were by injecting the beeswith concentrated solu- extracted in 10 ml water + 2 ml carbon tetrations of various proteins. Because of this,, the chloride. The extract was coarsely filtered. virus in apparent1.y healthy bees can be posi- centrifuged at 8000 K and the clear bacteriatively detected when acute paralysis is caused free supernatant fluid tested as follows. 167

168

BAILEY

( I ) One pl of decimal dilutions of the extract was injected, as described by Bailey et al. (1963)) into individuals of groups of bees. There were 20 bees per treated group and each group was kept in a small cage supplied with water and concentrated sucrose in gravity feeders. Each bee was injected with the equivalent of IO-” or less of a sample bee and was then incubated at 35°C. Bees that died between the 4th and 8th day were collected and stored at -2O’C. Those killed and paralyzed by the most dilute extract were extracted and decimal dilutions injected into groups of local bees,as before. This was done to tind the dilution end point of extracts containing freshly cultured virus because the samples were partly or wholly of bees that had been dead for an unknown period and CBPV losesinfectivity in dead bees kept at about 18°C (Bailey et al., 1963). (2) The incubation time of diseasein bees injected with serial dilutions of extracts was found in groups incubated at 35°C and 30°C. (3) The infectivities of extracts, diluted to give the equivalent of 10m5or less of a bee per injected dose, were tested after mixing them with equal volumes of either dilute ( ‘/{ ,,(, ) antiserum, prepared in rabbits against the virus (see Bailey et al., 1963; Bailey, 1965), or dilute normal rabbit serum. (4) The precipitates from extracts centrifuged at high speeds(see Bailey et al., 1963) were examined in the electron microscope. The live bees tested for ABPV came from Canada in May and Italy in June. A sample of local bees was also tested with them on each occasion. Ten bees of a sample were extracted in 1 ml of water + 1 ml of carbon tetrachloride and the emulsion was filtered and clarified as before. Each bee in half the remainder of the sample was injected with 1 ~1 of the extract and incubated at 30’ C. Under these conditions local bees always develop acute paralysis and die between about 4 and 7 days after injection. The beesin the other half of each sample were injected with water. Dead bees were collected daily from

the 4th to the 7th day and were preserved at -20” C. On the 7,th day the remaining live beeswere anesthetized and added to the dead of their group. Ten bees of each group were extracted in 10 ml of water + 2 ml of carbon tetrachloride. The extract was clarified as before and tests made as in (2), (3)) and (4) above. Antisera prepared against either ABPV or CBPV will neutralize much of the infectivity of the other virus, although the two have been shown to be serologically distinct by infectivity neutralization tests with absorbed antiserum (Bailey, 1965). It is laborious to prepare suitably absorbed antiserum, however, and its usein combination with the other tests was unnecessaryto identify the viruses. RESULTS

Chronic

Bee Paralysis

Virus

Local bees injected with the equivalent of lo-” to lOpi of a bee from each of the various sources became paralyzed after 5 or 6 days at 35°C. Bees injected with the equivalent of IO-’ to 10-n of these freshly paralyzed beesbecame similarly paralyzed. Paralysis and death was delayed by a day or two when bees were incubated at 30°C. Antiserum prepared against CBPV completely neutralized the infectivity of virus from all sources. Normal serum had no effect on infectivity. Electron microscopy showed many particles resembling CBPV in extracts of bees from all sourcestogether with fewer and variable numbers, sometimes almost none, of particles resembling ABPV. Acute

Bee Paralysis

Virus

All the imported live bees injected with concentrated extracts of their companions died by the 7th day, when few or none of those injected with water had died. Local bees injected with the equivalent of 10-a to 10W1’) of the killed imported bees became acutely paralyzed, showing disease much sooner at 30°C than at 35°C (Fig. 1). The extracts of the killed imported bees had their infec-

BEE-PARALYSIS

/: ‘/ / // I .’ I/ - 4/ 35°C

,/

/

/

3o”c

/ zy C J’

I

I

10-g DOSE

lo-

‘O

completely neutralized by antiserum against ABPV but not by normal serum, and electron microscopy showed they contained many particles resembling ABPV.

tivity

prepared

DISCUSSION

It is clear that all samplesof bees suffering from Waldtrachtkrankheit and Ma1 Noir were infected with as much CBPV as beessuffering paralysis

diagnosed

169

supports the claim (Bailey, 1965) that CBPV is the essential cause of these diseasesand that their different signsare causedby secondary factors. ABPV was present in the normal bees from Canada and Italy. The long incubation period at 35” compared with 30” in bees injected with terminally effective dilutions of ABPV was very striking compared with the previous results with concentrated extracts (Bailey, 1965) and was much greater than the opposite effect obtained with CBPV. This effect of temperature together with the dilution end point of extracts of freshly paralyzed bees seemthe easiestreliable methods of identifying and distinguishing between the two viruses. Thanks are expressed to Dr. S. F. Ruttner, Lunzam-See, Austria, and H. P. Wille, Liebefeld-Bern, Switzerland, for samples of bees with Waldtrachtkvmkheit; to Prof. Maria A. Vecchi, Bologna, Italy and Mr. 0. Rosenberg, Oslo, Norway for samples of live bees and bees with MaZ Noir; and to Dr. M. V. Smith, Guelph, Canada for live bees. Mr. R. D. Woods, Rothamsted Experimental Station, kindly did the electron microscopy and Dr. A. J. Gibbs prepared the antisera. REFERENCES

Fro. 1. The time taken for 10 out of 20 bees to die, at 30°C and 35’C, after each bee was injected with a dose of 10-a or 10-r” of the virus from an acutely paralyzed bee. Source of virus, -----, Italy; ---.-.-. ! Canada; --___. Britain.

from

VIRUSES

in

Britain.

This

L. 1965. Paralysis of the honey bee, Apis mellifera Linnaeus. J. Invertebrate Pathol., 7. 132-140. BAILEI’, L., AND GIBBS, A. J. 1964. Acute infection of bees with paralysis virus. J. Insect Pathol., 6, 395.407. BAIZEY, L., GIBBS, A. J., AND WOODS, R. D. 1963. Two viruses from adult honey bees (Apis mellifera Linnaeus) t’ivology, 21, 390-395. LOTMAR, R. 1940. Beobachtungen uber die “Morison’schen Zelleinschlusse” im Dunndarm. Landwirtsch. Jahrb. Schweiz., 54, 787-791. MORISOX, G. D. 1936. Bee paralysis. Rothamsted Conf., 22, 17-21. I’ECCHI, M. A., AND ZAMBONELLI, C. 1961. Recherches sur l’etiologie du “Ma1 Noir” des Abeilles (Apis nzellifica L.) en Italic. Bull. BpiCole, 4. 181-198 BAILEY,