AmppLu MicRomoLoGy, May 1972, p. 923-930Copyright ) 1972 American Society for Microbiology

Vol. 23, No. 5Printed in U.SA.

Mass Purification of Nucleopolyhedrosis VirusInclusion Bodies in the K-Series Centrifuge

J. P. BREILLAIT, J. N. BRANTLEY, H. M. MAZZONE, M. E. MARTIGNONI, J. E. FRANKLIN,AND N. G. ANDERSON

Molecular Anatomy Program and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee37830; Northeastern Forest Experiment Station, Hamden, Connecticut 06514; and Pacific Northwest Range

and Experiment Station, Corvallis, Oregon 97331

Received for publication 12 January 1972

Nucleopolyhedrosis virus inclusion bodies specific for Hemerocampa pseu-

dotsugata, Neodiprion sertifer, Porthetria dispar, and Heliothis zea have beenpurified by using a continuous-sample-flow-with-isopycnic-banding centrifugein quantities up to 6 x 1013 polyhedral inclusion bodies per day. Continuous-flow methods for S-p type purification have been evolved to deal with mass iso-lation of bioparticles.

The use of nucleopolyhedrosis viruses forbiological control of insects is under study inmany laboratories. Maximal use of the agentswill require maintenance of microbial contami-nants at safe levels and may require removalof the potent insect allergens present in some

species. For example, the hairs of the Douglas-fir tussock moth and the gypsy moth caterpil-lars can cause a severe contact dermatitis inhumans. These hairs are present in crudehomogenates, and if formed into an aerosol foraerial application, would constitute an obviousdanger to mammalian respiratory tracts.We have investigated the use of large-scale

density gradient centrifugation for the purifi-cation of the polyhedral inclusion bodies (PIB)of nucleopolyhedrosis viruses.The initial isolation of polyhedrosis virus

PIB by density gradient centrifugation was inswinging-bucket rotors (12). While this tech-nique remains unsurpassed for analytical pur-

poses, as a preparative method it is limited inthe quantity of product it yields.The batchwise use of the B-XXIII and the

B-XV zonal rotors resulted in the isolation of 3x 1011 pure PIB per run from Douglas-fir tus-sock moth larvae (Hemerocampa pseudotsu-gata) (13). We have since isolated PIB specificfor the corn earworm (Heliothis zea), the Euro-pean pine sawfly (Neodiprion sertifer), and thegypsy moth (Porthetria dispar) in similarquantities in the B-XXIX rotor (3). While thisrepresents a considerable gain over theamounts obtainable from the swinging-bucketrotor, the quantities isolated are still short ofthose required for field use of PIB as insectcontrol agents.

We therefore turned to large-scale contin-uous-sample-flow-with-isopycnic-banding cen-trifugation in the K-series system (4). A newrotor, K-X, designed for isolation of microm-eter-sized particles has been used successfullyto prepare pure PIB in quantities of 6 x 1013per run from the diseased larvae of each of thefour insect species previously mentioned. Pre-liminary results of this work have been re-ported (7).

Significant advances in the methodology ofcontinuous - sample - flow - with - isopycnic-banding centrifugation have been made inevolving the present technique for PIB isola-tion from larval homogenates.

MATERIALS AND METHODSSample preparation. The larval preparations

were: H. zea, Biotrol-VHZ, a lyophilized extractcontaining 3 x 1010 PIB/g, obtained from NutriliteProducts, Buena Park, Calif.

N. sertifer and P. dispar: diseased larvae werehomogenized in water and the suspension waspassed through cheesecloth. The material was thencentrifuged at low speed to remove large contami-nants. The resulting aqueous suspensions containedca. 2 x 109 and 5 x 10' PIB/ml, respectively. Analternate preparation from P. dispar was made bydisrupting diseased larvae and suspending them inwater. The material that settled to the bottom of thecontainer was collected, dried, and ground.

All subsequent procedures were carried out inmedia containing 0.01 M tris(hydroxymethyl)amino-methane-hydrochloride, 0.00075% dioctyl sodiumsulfosuccinate, pH 7.2-7.4 at 25 C (TD buffer). Allsucrose concentrations are expressed as percentw/w.

In the cases of H. zea, H. pseudotsugata, and thedried P. dispar, a 5% larval homogenate in 43%

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sucrose was prepared in a 1-gallon Waring Blendor attop speed for 2 min and then filtered throughcheesecloth.The N. sertifer and P. dispar aqueous suspensions

were diluted with 66% sucrose to a concentration of to yield43% sucrose and homogenized in the Blendor to en-sure dispersion of the PIB.

Centrifugation. An aluminum K-X rotor (J. P. 1Breillatt,- J. N. Brantley, and R. F. Gibson, manu-script in preparation) was operated in a K-seriescentrifuge with a C-type casing (4) equipped for where [temperature control of the rotor and fluid flow lines ters), pp(6). Sample suspensions were held in 20-liter pres- and mrnsure cans (model XX67-000-05 pressure tanks, Milli- flow strfpore Corp., Bedford, Mass.) with the sample flow and f/fnstream propelled by air pressure. The sample flow the nearrate was regulated by a throttle valve on the outflow and havfline and monitored with a calibrated flow meter or In proby sample collection. have bee

Gradients were loaded and recovered with the (16) forrotor at rest. The gradients were reoriented into the Accorditspin configuration by controlled acceleration (4): 0 to cleanout500 rev/min at 2 rev per min per sec, 500 to 2,000 that calcrev/min at 4 rev per min per sec. Sample flow was Quaninitiated at 2,000 rev/min, and the rotor was acceler- gradientated under full drive air pressure to the final ve- in a Petlocity. After exhaustion of sample, the rotor was op- Line henerated at 35,000 rev/min for the indicated time in- Microterval, to band the PIB, and the rotor was deceler- plate coated by the reverse of the controlled-acceleration and violprocedure. The gradients were recovered as 100-mlfractions through the virus peaks and as 400-ml frac-tions elsewhere. Consi

Gradient recovery was monitored at 580 nm by a initiallyspectrophotometer equipped with a flow cell and by aramemicroscopy inspection of the recovered fractions. parameThe fractions containing pure PIB were diluted 1:1 desiglwith TD buffer and centrifuged in a Spinco type 19 rates olrotor or passed through a K-XI rotor (J. P. Breillatt, mogenaR. M. VanFrank, R. F. Gibson, and J. N. Brantley, K-X romanuscript in preparation). The pellets were pooled TD bufJand washed once by centrifugation in TD buffer and rotor vethen resuspended in the same buffer. The less pure Onefractions from the sides of the PIB peak were carried nized irthrough the same procedure separately. a 10 to

Theoretical calculations. Fractional cleanout of at 24 liparticles from the sample flow stream was predicted analysisby equation 29 of Sartory (17): Most of

8=r3r2L (rev/min)2 dient, 1F

1016(1) large ai

cloggedwhere F is the fractional cleanout of the particle spe- debris icies of interest (p) and is defined as (Plnriux - to bandPoutflux)/Pinflux, r is the rotor core radius, L is the gelatinceffective rotor core length, Q is the sample flow rate PIB tre(liters/hour), and S is the sedimentation coefficient .u.of the particle (expressed in Svedberg units at the equilibasample flow stream temperature and composition). tion, a

Since the sedimentation coefficients of large bio- the coreparticles are not as commonly known as their diam- browneter and isopycnic density, S can be replaced in the rot(equation 1 by dient.

s D= ( PP Pm )1013

18 ilm(f/fmin)

47rSr2L DP2(pp - pm,) (rev/min) 2F = f

9 X 103 )7m(f/fmin) Q

(2)

(3)

)P is the diameter of the particle (centime-is the density of the solvated particle, Pm

are the density and viscosity of the sampleeam at operating temperature ('im in poises),nin is the frictional coefficient. In the case ofr-spherical PIB, we have set f/f0 equal to 1e neglected the hydration term.*actice these equations (equations 1 and 3)en found to be optimistic by a factor of 2 to 3possible reasons discussed by Sartory (17).ngly, after solving for a flow rate at 100%t, the flow rate used is one-half to one-thirdculated.titation. PIB concentration of sample andfractions was determined by particle countstroff-Hausser counting chamber or a Bright-mocytometer using phase-contrast optics.organisms were assayed by using standardunt agar (Difco) for total bacterial countsat red bile agar (Difco) for coliforms (1).

RESULTSiderations of purification cost led usy to seek rotor velocity and sample flowters applicable to a large centrifugein the 5,000 rev/min range with flow

If 100 to 200 liters/hr of 5% larval ho-Lte. As a test system, flow rates for thertor were calculated (5% homogenate infer at 25 C) to be 25 and 100 liters/hr atelocities of 5,000 and 10,000 rev/min.kilogram of Biotrol-VHZ was homoge-n 18 liters of TD buffer and passed over65% sucrose gradient in the K-X rotoriters/hr and 5,000 rev/min. Microscopys of the effluent showed removal off the PIB but, on recovery of the gra-the sectors unloaded unevenly due toLmounts of debris in the gradient thatthe rotor drain lines. Analysis of the

and the gradient showed this materialI isopycnically in 40% sucrose as a thickDus mass, thereby partially inhibitingansport through the gradient to theirrium position at 57% sucrose. In addi-lipid layer 3 mm thick had formed one surface, while a 5-mm layer of densedebris had built up on the outer wall of;or cavity, thereby displacing the gra-

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These initial results led us to seek condi-tions selectively to inhibit unwanted particleentry into the gradient. Efficiency of removalof a particle from the sample flow stream is adirect function of its sedimentation coeffi-cient; therefore, through manipulation of thesample stream flow rate and rotor velocity, wecan effectively cut off the entry into the gra-dient of particles with sedimentation coeffi-cients below a given value. By using two rotorsin cascade (Fig. 1), the first to remove un-wanted particles of high sedimentation coeffi-cient, the second to pass unwanted particles oflow sedimentation coefficient, only particleswithin a preselected sedimentation coefficientinterval will be captured in the gradient of thesecond rotor. The same effect can be achievedby sequentially performing the two separateoperations in a single rotor.The first rotor operates under conditions

such that 1% of the particles of interest (p,) areremoved from the flow stream. Then particleswith sedimentation coefficients 10 times thatof pi will be removed from the stream at 10%efficiency, and those particles with sedimenta-tion coefficients 100 times that of p, will becompletely removed from the sample stream.Thus the effluent from the first centrifugalstage (characterized by relatively low rotorvelocity and high sample flow rate) will con-tain 99% of the desired particles and very fewmore rapidly sedimenting contaminants. Thiseffluent is passed through the second rotorunder conditions of flow and rotor velocitysuch that 100% of the desired particles re-maining in the effluent from the first rotor arepulled into the gradient. Particles whose sedi-mentation coefficients are one-tenth that of piwill move into the gradient at .10% efficiency,while the particles leaving the flow stream at1% cleanout will have sedimentation coeffi-cients one-hundredth that of pi.One of the most powerful concepts in virus

and bioparticle isolation is the S-p separationscheme (2). Following a suggestion by CarlPrice (personal communication), we have de-parted from the usual form of the S-p plot,which employs the sedimentation coefficient ofthe particles corrected to water at 20 C, andhave used the sedimentation coefficient of theparticles in the sample medium. This permitsa more realistic judgment of the potential reso-lution of the separation process, primarily byemphasis of the flotation (negative sedimenta-tion coefficient) of particles less dense than thesample medium. When the results of the rotor-velocity-sample-flow-rate manipulations are

100%

[t

0%

-_ i199% ........

99n R

RPM: 3,500Q/3:300 I/hr

S5x 15x1055x1I6

RPM: 35,000Q/3: 30 I/hr

F

1%10%

100 %

S5 x le05 x 1025 x 101

F100%10%I1%

FIG. 1. Two continuous-sample-flow-with-iso-pycnic-banding rotors arranged in cascade to createa sedimentation coefficient interval between 5 x 1Osand 5 x 104 S in 43%o sucrose at 25 C. Particleswhose sedimentation coefficients fall in this rangewill be preferentially collected in the second rotor.Fractional cleanout values (F), sedimentation coeffi-cients (S), and the corrected volumetric flow rate(Q/3) are calculated from equations 1 and 2 for rep-resentative PIB.

plotted on the S-p plot (Fig. 2), a sedimenta-tion coefficient interval is observed withinwhich particles enter the gradient, there to befurther resolved by isopycnic banding. Whilethis does not constitute a single-step S-p pro-cedure, the end result, with kilogram quanti-ties of material, is practically identical withthat of such a procedure. A similar degree ofseparation efficiency is not currently feasibleby other methods.For the present case, this technique excludes

from the final purification gradient all large,dense debris and all lipids and small contami-

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SOLUBLE PROTEIN

K> POLIOeMOUSE ENCEPHALOMYOCARDITISe

# X 740 *FBACTERIOPH

RIVOOAL SUBIUNITS, RIB

RNA

_10 -b _ 1bl0o -10' 6lolo-l

APPL. MICROBIOL.

SEDIMENTATION COEFFICIENT AT 25' IN 43 % w/w SUCROSE (Svedbwrg units)

FIG. 2. S-p separations plot showing the selectivity achieved by combining the sedimentation coefficientinterval with sample density inhibition of particle transport into the gradient for purification of PIB fromsubcellular components. Particles with density less than the sample density, 1.19 g/cm3, cannot sedimentfrom the sample flow stream as shown by the negative sedimentation coefficient (flotation coefficient). Ver-tical lines denote percent cleanout of particles from the sample flow stream under conditions of Fig. 1.

nating particles, which together comprise thebulk of the undesirable particles in the larvalhomogenate.These considerations require a sample flow

rate of 300 liters/hr in the first rotor (Fig. 1and 2). However, flow rates above 50 to 60liters/hr are not satisfactorily obtained in thepresent system under the conditions used. Atthe maximum flow rate used (48 liters/hr), the1% cleanout level corrected for nonideal flowconditions in the sample stream is at 7 x 103S (PIB of 1.0 gm diameter), and the 100%cleanout level is at 7 x 105 S (PIB of 10 ,umdiameter). Due to this lower sample flow rate,more of the PIB population is collected in thefirst rotor than originally intended in the theo-retical design (Fig. 2). However, this departurefrom the theoretically derived procedure is oflittle import, since the PIB collected in thefirst rotor are recovered from the gradient, di-luted, and added to the sample flow stream ofthe second rotor before flow through thesecond rotor is completed. Indeed it is withthe first rotor that one has the greatestfreedom to vary the run parameters to selec-tively avoid more rapidly sedimenting contam-inants.The other source of interfering material in

the sample was the substance that banded atabout 40% sucrose and inhibited gradient re-

covery and inclusion body transport within thegradient. It was excluded from the gradient byadding sucrose to the homogenate until thesample density equalled the isopycnic banding

density of the interfering particle. This causedthe particle to remain in the sample streamand permitted the gradient to be designedsolely for high-resolution separation of PIBfrom bacteria, spores, and other particles ofsimilar size and density present in the larvalhomogenate.

Because the rate of removal of PIB from theflow stream (and thereby the time-cost rela-tionship of purification of the inclusion bodies)is related to the density and viscosity of thesample flow stream, the density of the samplehomogenate was increased just to the levelthat would exclude the unwanted particles,i.e., 43% sucrose. This maximized the flow rateachievable under conditions that minimizedthe appearance of this specific contaminant inthe gradient; nevertheless, the sedimentationrate of the inclusion bodies was reduced 22-fold. For this technique, a density-creatingsolute with negligible viscosity might be pref-erable to sucrose, regarding the rate of particleremoval from the flow stream; however, if su-

crose were still used for the gradient, at thesedensities an appreciable cross-diffusion of gra-dient solute and sample density solute (if dif-ferent) would occur, creating a complicatedsituation with respect to particle transport andpressure drop along the sample flow stream.The increase in sample density and viscosity

necessitated an increase in rotor velocity to25,000 rev/min to maintain 100% cleanout ofthe inclusion bodies at 25 liters/hr. Thus theK-X rotor, which was to be a test system for a

926

MITOCHONDRIA

1.0-

I.2 -

F 14-

z

al

1 8-

2.0-104

PARTICLE CLEANOUTSECOND ROTOR FIRST ROTOR

1% 10% 100% 1% 10% 100%

NUCLEOPOLYHEDROSIS VOWS/ NCLUSION KOWES

RIFT VALLEY FEVER DIAMETERA(pm K

POLYOMA X ' B 12* ')ADENOV1RUS 2

SNOPE PLLOMA NUCLEI/@ REOVIRUS 3

FOOT AND MOUTH1AGES T3Ti

v

BOSOMES, AND POLYSOMES

GLYCOGENl0 0| |DNA~~~~~~~~~~~~~~~

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conceptual large, slow rotor, now approachesthe optimal-design rotor for this separationdue to the presence in the larval homogenateof an interfering particle. It is possible thatthis interfering substance occurring in H. zeapreparations is not present in all species, thuspermitting the use of more efficient condi-tions.To evaluate the gradient requirements, 250 g

of Biotrol-VHZ was homogenized in 4.75 litersof 43% sucrose and passed over a 47 to 65%sucrose gradient in a K-X rotor at 15,000rev/min, 11 liters/hr, 25 C. The interferingmaterial did not enter the gradient. The PIBbanded as a single peak. However, on micro-scopy inspection of the peak fractions, nu-merous dead yeast cells and other materialwere observed. The peak fractions were pooled,pelleted, and then isopycnically banded in aB-XXIX rotor containing a 45 to 62% sucrosegradient. Four peaks were resolved, with deadyeast cells banding at 60 and 58.2% sucrose,the inclusion bodies at 56.8% sucrose, and mis-cellaneous debris below 50% sucrose.A gradient was designed to exclude the con-

taminating particles based on these data andthe following rationale. The banding positionsof unwanted particles (58.2 and 50% sucrose)should be far from the isopycnic position of theinclusion bodies (56.8% sucrose). The PIBbanding zone should be a broad flat region inthe gradient to allow maximum capacity forPIB banding without formation of a thicklypacked zone that would block passage of den-ser, but more slowly sedimenting, contami-nants. A stacked gradient was chosen for easeof operation and because it closely approachedthe ideal gradient; however, once the gradientis formed in the rotor, diffusion rapidly oblit-erates any sharp steps (Fig. 3).

6~5

5-__.___ -60

I ~~~~~~~~~~~~~~~55(

-50.

VOLUME (liters)

FIG. 3. Isolation of PIB from N. sertifer larvae ona step gradient in a K-X rotor. -, Initial gradient; 0,

recovered gradient.

The gradient was tested by passing a 25-literpreparation from N. sertifer (2 x 10'3 PfIB)through the rotor at 25 liters/hr and 30,000rev/min. The first pass through the low-ve-locity rotor was neglected in this case becauseof the prior removal of large debris duringsample preparation. PIB cleanout efficienciesof 85% per pass through the rotor were ob-tained with 97% cumulative recovery of PIB byrecycling the effluent through the rotor (14).The PIB were well resolved from debris andcontaminants, as shown by the gradient profile(Fig. 3) and by microscopy analysis of the re-covered peak fractions which had the macro-scopic appearance of a creamy white slurry. Insubsequent runs, quantities up to 6 x 1013 PIBhave been processed in a single rotor.To test the entire system, 1.2 kg (wet weight)

of diseased H. pseudotsugata larvae was pre-pared and passed through a K-X rotor oper-ating at 3,500 rev/min with flow rates of 42 to48 liters/hr. The rotor contained a step gra-dient of 2 liters of 60% sucrose and 4.7 liters of47% sucrose. After termination of flow therotor was operated at 30,000 rev/min for 15min to permit the large particles to reach equi-librium. The fractions containing PIB wererecovered from the rotor, pooled, diluted withTD buffer to 43% sucrose, and added back tothe effluent. This effluent was then passedthrough a K-X rotor at 24 liters/hr, 30,000rev/min, 22 C, over the derived gradient (Fig.4A). After exhaustion of the sample, the rotorwas operated at 35,000 rev/min for 70 min tobring the larger particles to their isopycnicdensity. These field-collected larvae sufferedfrom a mixed infection, as shown by the cyto-plasmic polyhedrosis inclusion body peak at 1liter gradient volume. Microbiological assay ofthe recovered fractions showed that certain ofthe microorganisms banded isopycnically inthe same density range as the PIB (Fig. 4B).When the dried P. dispar larvae preparation

was processed by a two-step procedure in theK-X rotor (in the same manner as the H. pseu-dotsugata), upon recovery of the gradient fromthe second rotor all fractions contained visibledebris. This was traced to dense fragmentsinsufficiently solvated to stick to the wall ofthe first rotor. When the gradient was re-covered from the first rotor (at rest), the debriswashed off the wall into the various fractions;when the fractions containing PIB were addedback to the sample for the second rotor, theprocess repeated itself. The PIB from that par-ticular run were pooled and banded in a B-XVrotor and then dynamically unloaded to re-move the contamination. Subsequent P. dispar

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C EE 0 3- . -

2 1 2 3 4 5 6 ,50

F- 40

0~~~~~~~~~~~

0~~~~~~~~~~~~4~E32-S32N

4~24- 24

l16ab16aa(r~~~~~I

0 ~~~~04~< 8- f-\ 8 -J0 0 ~~~~~~~~~~0U

createb equnilueoaA-K-X roo.()Ga

sO 4

0 2 3 4 5 6 7VOLUME (liters)

FIG. 4. Isolation of PIB from H. pseudotsugatalarvae by using a sedimentation coefficient intervalcreated by sequential use of a K-X rotor. (A) Gra-dient profile from second rotor; 0, recovered gra-dient. (B) Bacteria distribution in fractions re-covered from second rotor.

samples were prepared as the aqueous suspen-sion. This solvated the debris to the extentthat it adhered to the K-X rotor wall duringthe unloading procedure (Fig. 5A) and re-moved the need for the centrifugation step inthe B-XV rotor.As seen in Fig. 4B for H. pseudotsugata and

in Fig. 5B for P. dispar, microorganisms arefound in higher concentrations in the PIBbanding density range (possibly spores whosesedimentation was retarded by the tightlypacked PIB in the zone) and also at the pe-riphery of the rotor (gradient volume of 0 to 1liter). By analogy to the debris problem, onewould expect contamination of all fractions bypelleted bacteria or spores to the extent thatthey were washed off the wall during staticgradient recovery. Dynamic gradient un-loading could avoid this problem, but a high-resolution method to unload dynamically acontinuous-sample-flow K-series rotor has notyet been devised.

While satisfactory results were obtained withPIB isolations from N. sertifer, P. dispar, andH. pseudotsugata larvae in the amounts availa-ble, a large-scale isolation from a debris-ladensample was necessary to investigate the ca-pacity of the cascaded K-X rotor system. Twokilograms of Biotrol-VHZ (6 x 1013 PIB) was

processed through two K-X rotors in cascade:the first operated at 3,500 rev/min with asample flow rate of 42 to 48 liters/hr, thesecond operated at 35,000 rev/min with asample flow rate of 20 to 24 liters/hr. Thesample was passed through the second rotortwice, with approximately 80% cleanout of PIBper pass through this rotor as judged by hemo-cytometer counts on the sample and effluents.During this run, lipid-containing material ac-cumulated in the inflow lines of the rotor andwas prevented from entering the rotor cavityby the density difference of the material andits suspending medium, 43% sucrose. In thiscase the pp - Pm term (equation 2) produced anegative sedimentation coefficient (a flotationterm) for the lipid-containing particles, whichaccumulated in the lines and effectivelyblocked the sample flow. The lipid block wasperiodically removed by back-flushing thelines. This recovered material was extractedwith methanol-chloroform and yielded a denseprecipitate, easily sedimentable in 45% su-crose, plus a yellow solution. A lipid solventthat does not inactivate the PIB or other pre-liminary steps to remove the lipid would solvethis problem, since a suitable modification ofthe rotor is not readily apparent.

E

c

E0

0

44

E

co

S28

v)0

0

cr

a:

0-

0-)j-

'E

m

or

VOLUME (liters)

FIG. 5. Isolation of PIB from P. dispar by using asedimentation coefficient interval created by sequen-tial use of a K-X rotor. (A) Gradient profile fromsecond rotor. -, Initial gradient; 0, recovered gra-dient. (B) Bacteria distribution in fractions re-covered from second rotor.

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Microbiological assay of the pooled PIBpreparations (Table 1) yielded the followingconclusions. Significant reductions in bacterialcounts were achieved only with N. sertiferwhich had been precleaned and with H. zea

which was produced in a controlled insectary,suggesting that the strains of bacteria thatband in the same density range as the PIBmay be species-specific, removed by the pre-

cleaning step, or absent from the insectaryenvironment.The infectivity of the inclusion bodies in

their respective hosts was not changed by thecentrifugal purification process.

DISCUSSIONThe inclusion bodies purified by this method

are free from insect components and debris on

microscopy inspection. The significance ofthis fact for studies on inclusion body proteinand virus components is evident. The signifi-cance of the fact for use of the inclusion bodiesas a biological control agent is questioned byseveral studies which show that crude andsemipurified preparations of nucleopolyhedro-sis virus inclusion bodies from H. zea andTrichoplusia ni are nontoxic and nonpatho-genic in the guinea pig and only mildly aller-genic in this animal (9, 10, 15). A commonfinding in the two studies on H. zea was that aguinea pig from the inhalation test group diedfrom pneumonia after exposure to the prepara-tion, which seems to preclude allergenicity butmay indicate a primary reaction to the agent.The possible significance of this occurrencemay be found in considering H. pseudotsugatalarvae, whose airborne hairs cause a mild tosevere contact dermatitis to loggers andfarmers working in the woods during an out-break of the insect. In studies with rabbits,crude preparations of H. pseudotsugata werenonallergenic but caused a minor primary skinirritation (18). This raises the question ofwhether guinea pigs may have an individualprimary response to these agents, as do hu-mans, and emphasizes that animal reaction to

a larval preparation must be considered sepa-rately for each insect species.The reaction of animals to N. sertifer inclu-

sion bodies purified by this method and to thecrude starting material is being assayed by anindependent testing company for the North-eastern Forest Experimental Station of theForest Service (U.S. Department of Agricul-ture). To date no pathogenicity, toxicity, orallergenicity has been found in a number ofstandard test procedures (F. B. Lewis, personalcommunication).However, the responses of large sectors of

the human population repeatedly exposed tolarge quantities of airborne insect dusts are notknown in detail. In addition to dermatitis,cases of conjunctivitis and asthma have beenreported in human beings after exposure tobody parts of various arthropods, even as dustsof dead insects. Furthermore, the intended oraccidental contamination of food crops withproducts containing insect fragments may leadto an excess of the tolerances for filth of insectorigin set by the Federal Food, Drug, andCosmetic Act. The origin of insect fragmentcontamination can be traced by means of re-fined methods of sanitation-analytical ento-mology (11).Even though the medical and legal implica-

tions of large-scale field applications of crudepreparations have not yet been fully evaluated,it would seem cautious and advisable to striveto reduce the insecticidally inactive bulk(mostly insect debris) of nucleopolyhedrosisvirus preparations.The reduction of bacteria in the PIB prepa-

rations purified by the present technique wasnot as great as that accomplished by the two-step batch procedure in the B-series rotors(13). This may be due to several factors pre-viously discussed herein, primarily that ofstatic versus dynamic gradient recovery. Thecounts reported for coliform bacteria werethose present on the violet red bile agar; how-ever, we suspect that these may not trulyrepresent enteric bacilli because of the gray

TABLE 1. Bacterial contamination of homogenate and purified PIB

Homogenate Pooled peak fractions

Species Total aerobic Coliform Total aerobic Coliformbacteria per bacteria per bacteria per bacteria per

109 PIB 109 PIB 10'PIB 109 PIB

Hemerocampapseudotsugata ................. 3.7 x 107 9.1 X 105 1.7 x 106 7 x 103Neodiprion sertifer ............ .............. 4.0 x 108 2.5 x 103Porthetria dispar ............... ............. 1.5 x 107 7.7 x 105 1.2 x 106 5.1 x 104Heliothis zea.5.9 x 102 7.1 x 104 2.1 x 101

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930 BREILLAI

coloration of the colonies. Several unsuccessfulattempts were made to identify the predomi-nant species. During the preparation of thismanuscript, Cline et al. (8) have reported thatdebris and bacterial contamination of zonallypurified PIB can be markedly reduced by al-lowing bacterial digestion of the larval homog-enate starting sample, as used by Bergold (5).This is followed by an overgrowth of a bacillus,thus necessitating resolution of the PIB fromonly a single microorganism.

This appears to be the method of choice forfuture PIB purification schemes employing thezonal centrifuge, because it also digests thosesubstances which reduced the efficiency of thecontinuous-flow centrifugation step.

High-resolution separations in the zonal cen-trifuge are usually synonymous with narrow,low-capacity zones. We have attempted tocreate conditions in which very large amountsof product (PIB) can be separated, with rela-tively high resolution, from contaminants (bac-teria and spores) differing only slightly in den-sity. This has resulted in unusually wide,thickly packed peaks in shallow density gra-dients. To permit the use of these gradients, ithas been necessary to exclude the bulk of thecontaminants from the gradient through ma-nipulation of the rotor velocity and sampledensity and flow rate.The concepts of a sedimentation coefficient

interval and sample density inhibition of par-ticle transport into the gradient are generallyapplicable to all bioparticle separations in con-tinuous-sample-flow rotors. They greatly in-crease the capacity and efficiency of the K-se-ries centrifuge and are particularly useful forpreparations containing large volumes of un-wanted particles. Their use should permitemploying much cruder preparations as sam-ples than those currently used, since pre-cleanup procedures on volumes compatiblewith use of the K-series centrifuges are un-wieldy.

ACKNOWLEDGMENTS

We express our appreciation to John Carnegie for his in-terest and assistance with the purification of the H. pseu-dotsugata PIB, to Carl S. Rehnborg for providing samples ofBiotrol-VHZ, and to Franklin B. Lewis for his continuedinterest and preliminary information on test results.

The Molecular Anatomy (MAN) Program is supported bythe National Cancer Institute, the National Institute ofGeneral Medical Sciences, the National Institute of Allergyand Infectious Diseases, and the U.S. Atomic Energy Com-mission. Oak Ridge National Laboratory is operated byUnion Carbide Corporation Nuclear Division for the U.S.Atomic Energy Commission.

rnr ET AL. APPL. MICROBIOL.

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14. Mazzone, H. M., J. P. Breillatt, and N. G. Anderson.1970. Zonal rotor purification and properties of a nu-clear polyhedrosis virus of the European pine sawfly(Neodiprion sertifer, Geoffroy). Proc. IVth Int. Colloq.Insect Pathol., 25-28 August 1970, College Park, Md.

15. Meinecke, C. F., W. C. McLane, and C. S. Rehnborg.1970. Inhalation and dermal allergenicity studies of anuclear-polyhedrosis virus of Heliothis zea in guineapigs. J. Invertebr. Pathol. 15:207-210.

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