APP~En MIcRoaIouoGy, Jan. 1973, P. 115-122.Copyright 0 1973 American Society for Microbiology

Vol. 25, No. 1Printed in U.S.A.

Effects of Some Disinfectants on African SwineFever Virus

S. S. STONE AND W. R. HESS

Plum Island Animal Disease Laboratory, Greenport, Long Island, New York 11944

Received for publication 13 July 1972

Ten commercially available disinfectants were tested at high pH in 2%sodium hydroxide and low pH in 2% acetic acid as inactivants for African swinefever (ASF) in a protein-rich blood-spleen homogenate. As assayed in leukocytecultures, sodium hydroxide and acetic acid, sodium meta silicate and Roccaldid not inactivate ASF virus in 1 hr at 22 to 25 C. Some viricidal activity as

assayed in leukocyte cultures was found with Weladol, Triton X-100 Amphyl,pHisoHex, sodium dodecyl sulfate, LpH, Environ, Environ D, and One-StrokeEnviron. Of these, the last four appeared to be most promising. When assayed inpigs, only One-Stroke Environ (1/E) was viricidal. Concentrations of 1.0, 0.75,and 0.5 were effective, but, at 0.25%, virus was not inactivated. The minimaltime to inactivate ASF virus by 1% 1/E is 60 min. A room contaminated withASF virus was made safe for pigs after 1 hr by spraying with 1% 1/E. The mostactive component of 1/E is o-phenylphenol. Although another component of1/E, i.e., o-benzyl-p-chlorophenol, also has some activity, the mixture of theactive components of 1/E is most effective against ASF virus. One of the solubleantigens associated with ASF virus is destroyed by 1/E.

African swine fever (ASF) is no longer anobscure disease of Africa. Since 1957 it has oc-curred in Portugal, Spain, France, Italy and-inJune of 1971-Cuba (13). The causative agentis a deoxyribonucleic acid virus (1) thatis sensitive to ether and chloroform (5). Since asafe and effective vaccine has not been pro-duced, control of the disease depends entirelyupon rapid diagnosis, slaughter of infected andexposed animals, and strict imposition of effec-tive sanitary measures. This so-called "stamp-ing out" procedure was successfully applied inFrance and Italy, and the disease was elimi-nated before extensive spread occurred. How-ever, there is no published evidence that themeasures applied would be adequate or feasiblein areas having an intensive swine industry.

It is known that ASF virus may remainviable for long periods of time in feces, blood,and soil and on wooden surfaces (9). It is alsoknown that the virus suspended in a men-struum rich in protein may withstand ratherextensive variations in pH (5). Furthermore,proteolytic enzymes have little or no effect onthe virus (5). It is therefore likely that solutionscommonly used to disinfect premises whereanimal diseases have occurred are not com-pletely effective in destroying ASF virus; nor

will a waiting period of several months assurethat the premises are safe for restocking.

Laboratory methods for inactivation of ASFvirus have been reported (16). The methodsemploy fast-acting agents such as beta-propio-lactone, acetylethylenemine, and glycidalde-hyde. Even at low concentrations, these com-pounds are effective ASF virus inactivants, but,because of their instability in aqueous solu-tions, they are not suitable for use in the fieldwhere spraying affords the only practicalmeans of covering the large areas that must bedisinfected following an outbreak.The presence of extraneous organic matter

may greatly reduce the effectiveness of someclasses of chemical disinfectants. This is espe-cially true of chlorine compounds and cationicdetergents (17). A high concentration of or-ganic matter is a feature common to theenvironments most likely to be contaminatedwith ASF virus during an outbreak of thedisease. Since the work reported here wasundertaken in an effort to find a disinfectantcapable of rendering an infected premise safefor early restocking after an outbreak, thedisinfectants were tested against ASF virus inthe presence of a high concentration of pro-teins, and one agent that was effective under

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STONE AND HESS

these conditions was further used to decontam-inate a room containing several days' accumu-lation of litter, feces, and urine of pigs that diedof ASF.

MATERIALS AND METHODSVirus. The Lisbon strain of ASF virus (11) was

used. The stock virus suspension was prepared by I.C. Pan of this laboratory. Pigs infected by theintravenous route died of acute ASF 5 to 6 days later.Spleens were removed, homogenized in an equalvolume of whole defibrinated blood, filtered throughseveral layers of cotton gauze mesh, divided intoseveral portions, and stored in a dry-ice chest. Thestock virus suspension had a protein content of 21.3%as determined by micro-Kjeldahl analysis. A freshsample was thawed for each experiment. Hemad-sorption titers determined at the time of each experi-ment ranged from 101 to 107/ml.

Virus assay. In all inactivation experiments, thestock virus and virus-inactivant mixtures were ti-trated by the hemadsorption reaction (10) in pigleukocyte cultures (LC) prepared as described else-where (7). Titers were reported as log of 50% hemad-sorption per milliliter. The toxicities of the inacti-vants alone were simultaneously determined insimilar cultures.

Soluble antigens. Monolayer cultures of VEROcells were infected with ASF virus (5). When thecytopathic changes were well advanced, the cellswere suspended by shaking and were collected bycentrifugation. One volume of packed cells wassuspended in two volumes of phosphate-bufferedsaline and sonically disrupted at 0 to 5 C, using ten10-sec bursts of energy. The resulting suspension wasclarified by centrifugation at 10,000 x g for 30 min,and the supernatant fraction was used as antigen.Agar gel diffusion precipitation tests. Plates

were prepared with 0.85% Ionagar in 0.05 M boratebuffer, pH 8.6. The distance between the reagentwells was 3 mm. ASF survivor pig serum was used asan antibody source. Normal pig serum and an extractof sonically disrupted, noninfected cultured cellswere used as controls.

Immunoelectrophoresis. The microtechnique(14) was employed with 0.025 M barbital acetatebuffer, pH 8.6, and a constant current of 2 ma/cm for90 min. A 0.1% alcoholic solution of bromophenolblue was used as an indicator of electrophoreticmigration. The precipitin patterns were developedwith ASF survivor pig serum.

Inactivants. The sources of inactivants used wereas follows: LpH, Environ, Environ D, and One-Stroke Environ-Vestal Laboratories, Division of W.R. Grace & Co., St. Louis, Mo.; Weladol disinfect-ant-Allied Laboratories, Inc., Division of Pitman-Moore Co., Indianapolis, Ind.; pHisoHex-WinthropLaboratories, New York, N.Y.; Triton X-100 lotC11-Packard Instrument Co., Inc., Downers Grove,Ill.; Solventol -Soventol Chemical Products, Romu-lus, Mich.; sodium dodecyl sulfate (SDS) USPand sodium meta silicate (tech. grade), lot783701-Fisher Scientific Co., Fairlawn, N.J.; o-

phenylphenol, lot 711A, and p-tertiary-amylphenol,lot 8A-Eastman Organic Chemicals, Rochester,N.Y.; o-benzyl-p-chlorophenol-K & K Laboratories,Inc., Plainview, N.Y.

In addition to the commercially available germi-cidal agents listed above, 2% acetic acid and 2%sodium hydroxide were tested in the initial screeningexperiments, for these solutions have been widelyused as viricidal agents (3).Each of the inactivants was made up as a 1%

solution in both 2% acetic acid and sodium hydrox-ide. In later experiments, distilled or tap water wereused as diluents to prepare 1% solutions and theconcentration of the inactivant was altered as shownin the tables.

Tests in animals. Adult pigs weighing from 100 to200 kg were used in the ultimate determinations ofvirus inactivation. They were housed in rooms de-signed for maintenance of strict isolation (2), but,because of space limitations, only the pigs serving asvirus controls were housed separately. The pigsreceiving the virus-inactivant mixtures were housedtogether. All inoculations were by the intramuscularroute. Temperatures were taken daily, and a rise to103 F (39.4 C) or higher was regarded as the first signof ASF infection. The first animal displaying thefebrile reaction was regarded as having been infectedby the inoculum given. Any other animal in the roomthat became febrile within the next 2 days waslikewise considered to have been infected by theinoculum administered. If an animal's first febrileresponse occurred more than 2 days after that of thefirst reactor in the room, it was regarded as a possiblecontact infection. In any case, the delayed responsewould be indicative of either complete or substantialinactiviation of the virus in the inoculum given.Further, comparison of the day of fever or deathamong these pigs and the control pigs served as anadditional basis for analysis of the test results.

Spleen and kidney samples were taken from one ormore pigs in each experiment and assayed for ASFvirus by the hemadsorption test in LC. Tests werealso made for the presence of soluble antigen by usingagar gel diffusion precipitin reaction (6).

Inactivant screening test. The purpose of thistest was to eliminate any inactivant that would notreduce the titer of the test virus at least from 106 to103 in 60 min at room temperature (22-25 C). Onevolume of the stock virus suspension was mixed withnine volumes of the test compound. After 60 min, 1ml of the mixture was diluted 1:10 in culture fluidand the pH was adjusted to 7 to 8, by using 1 N NaOHor 1 N HCl. A 1-ml amount of the adjusted mixturewas further diluted 1:10 in the culture medium, andfour LC in short Leighton tubes were each inoculatedwith 1 ml of this dilution. In all cases, the pH of thissecond dilution of the virus-inactivant mixture was7.3 to 7.4 as measured by a glass electrode. Each ofthe test inactivants was treated in the same way andassayed for toxicity. The LC were examined daily forsigns of virus hemadsorption or toxicity.Assay of inactivated ASF virus in pigs. In the

first of these experiments, four of the most promisinginactivants, i.e., SDS, Environ, Environ-D, and

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One-Stroke Environ (1/E) were used at the 1% levelwith stock virus and a 60-min inactivation time. Ninevolumes of inactivant were mixed with 1 ml of virus.After 60 min, pigs were inoculated with the virus-inactivant mixtures.Minimum effective concentration. The second

experiment with pigs was to determine the minimalconcentration of disinfectant required to render thevirus noninfective for pigs. A 1-ml amount of stockvirus was mixed with nine volumes of 1.0, 0.75, 0.5,and 0.25% 1/E, and 60 min later two pigs wereinoculated with each of the virus-inactivant mixturesand assayed. Two pigs housed in a separate roomserved as virus controls.

Time of inactivation. The third experiment withpigs was designed to determine the minimal timerequired to inactivate ASF virus by 1/E. Nine vol-umes of 1/E solution were mixed with one volume ofstock virus suspension, and a 5-ml sample wasremoved at 15, 30, 60, and 120 min. Two pigs wereimmediately inoculated at each time interval withthe virus-inactivant mixture, and the virus wastitrated in LC at the same time. Two virus controlpigs were housed separately.

Transmission and disinfection experiment. Toestablish that an ASF virus-contaminated area couldbe effectively disinfected, it was first necessary toshow that the contaminant in the room could belethal to pigs. Accordingly, a room housing ASFvirus-infected pigs was not cleaned for 2 days. Duringthis time, two pigs previously infected with ASFvirus died of the disease. These pigs were removedand 4 hr later two susceptible pigs were placed in theroom. One pig developed a fever in 3 days and died onthe 9th day. The other pig appeared normal duringthis interval and was destroyed on the 10th day. Bothpigs were removed, and the room was thoroughlysprayed with a 1% solution of 1/E prepared in tapwater. All residual fecal material and feed weredispersed with 1/E. One hour later, four susceptiblepigs were placed in the room.

Viricidal activity of the active components ofl/E. Each of the three active ingredients in 1/E, i.e.,o-phenylphenol, o-benzyl-p-chlorophenol, and p-ter-tiary-amylphenol, was prepared at the same concen-tration and pH as found in a 1% aqueous solution of1/E. Each was tested singly and in combination, byusing the stock virus. Assay for virus in thesemixtures was in LC and in pigs.

RESULTSA summary of the results of the first screen-

ing test with 1% solutions of 10 disinfectants inboth 2% NaOH and acetic acid as well as thelatter reagents alone is shown in Table 1. Viruswas detected by the hemadsorption reaction inLC cultures in both NaOH and acetic acid,sodium meta silicate at both high and low pH,Roccal in the alkaline, and Solvental in theacid solutions. With the other disinfectants,i.e., LpH, Environ D, Environ, Weladol, pHi-soHex, Triton X-100, SDS, and Amphyl, there

was either no hemadsorption or the test was notreadable because of various degrees of cellulardegeneration caused by the disinfectant.The results of a second screening test em-

ploying disinfectants that appeared to be viri-cidal or toxic (Table 1) are shown in Table 2.These disinfectants were used as 0.1% aqueoussolutions, and the virus-inactivant mixture wasassayed in LC at the 102, 10- 3, and 10-4dilutions. Four of the nine disinfectants tested,Environ D, Environ, 1/E, and SDS, appearedto inactivate ASF virus at the 10-2 dilution. Incontrast, hemadsorption occurred in the 10-2dilutions of the LPH, Weladol, pHisoHex,Amphyl, and Triton X-100 virus mixtures.The three Environs and SDS were tested as

ASF virus inactivants as 1.0 and 0.25% aqueoussolutions. Pigs were used as the virus indicatorto circumvent the cytotoxicity of the disinfect-ants in LC at this concentration. Table 3shows the results of this test. Only the piginoculated with the 1% 1/E-virus mixture failedto develop ASF from the inoculum. The piginoculated with the 0.25% 1/E-virus mixturedeveloped ASF as did the pigs inoculated withSDS, Environ, and Environ D-treated virussuspension.

In determining the minimum effective con-centration of 1/E, each virus-inactivant mix-ture was tested in two pigs. As indicated inTable 4, the two pigs inoculated with the 0.25%1/E-virus mixture developed ASF and died intimes comparable to the virus control animals.The pigs inoculated with the 0.50, 0.75, and 1%1/E-virus mixtures had reactions indicative ofinfection by contact. It was therefore indicatedthat 1/E in concentrations of 0.5% and higherwas able to inactivate the stock virus suspen-sion.The results of the experiment to determine

the minimal time to inactivate the stock ASFvirus suspension are shown in Table 5. One ofthe two pigs (pig no. 21) inoculated after 15 minof inactivation developed ASF and died.Another animal (pig no. 23) that had receivedstock virus exposed to the disinfectant for 30min reacted within 2 days after the initialreaction of pig no. 21. Complete inactivation ofthe stock virus at 30 min was therefore re-garded as uncertain. However, inactivation at60 and 120 min was unequivocal, and, com-pared to the virus controls, it was evident thatsubstantial inactivation also occurred in thelesser time intervals.One of the two pigs housed in the con-

taminated room developed a febrile response 6days later and died on the 9th day. ASF virus

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TABLE 1. Preliminary screening of various disinfectants at 1% concentration in acid and alkaline solutions as

potential African swine fever viricidesa

Detection ofvirus in"

Disinfectant Active ingredients (concn as %)Acid Alkaline

solution solution

Amphyl

Environ

Environ D

One-Stroke Environ

LpH

pHisoHex

Roccal

Sodium meta silicate

Solventol

Triton X-100

Weladol

ControlAcid solutionAlkaline solution

Potassium ricinoleateo-Phenylphenolp-tert-AmylphenolAlcohol

o-Phenylphenolo-Benzyl-p-chlorophenolp-tert-Amylphenol

o-Phenylphenolo-Berzyl-p-chlorophenolp-tert-Amylphenol2, 2'-Methylene-bis (3-, 4-, 6-trichlorophenol)

o-Phenylphenolo-Benzyl-p-chlorophenolp-tert-Amylphenol

Glycolic acido-Benzyl-p-chlorophenolp-tert-Amylphenol

Hexachlorophene

Alkyl (C8 to C18 groups)Dimethyl-benzyl ammonium chlorides (Benzalkonium

chloride)

PolyphosphateSilicate as SiO2OrthophosphateSodium carboxy methylcellulose

Aralkyl polyether alcohol

Polyethoxy polypropoxy-ethanol-iodine complexNonyl phenoxy-polyethoxy-ethanol-iodine complexHydrogen chlorideComposition3% Acetic acid2% Acetic acid

(44.0)(15.0)(6.3)(4.7)

(3.9)(3.3)(0.8)

(4.6)(5.0)(4.5)(0.4)

(10.0)(8.5)(2.0)

(12.6)(6.4)(0.5)

(3.0)

(10.0)

(22.0)(2.2-4.4)(5.0)(1.0)

(100.0)

(7.9)(7.6)(0.1)

D

D

D

+

D

D

D

D

D

D

D

D

D

+

a Test procedure: 1% solutions of each disinfectant were made in 2% acetic acid and 2% sodium hydroxide.Stock virus (a blood-spleen mixture containing 106.5 hemadsorbing units of ASF virus per ml) was diluted 10xin the disinfectant solution and held for 1 hr at room temperature (22-25 C). The mixture was then diluted10 x in culture medium and adjusted to pH 7 to 8 with sodium hydroxide or hydrochloric acid. After another10x dilution in culture medium, 1.0 ml was put on each of four leukocyte cultures. Duplicate tests withoutvirus served as toxicity controls.

h +, Positive hemadsorption reaction indicating presence of active virus; -, negative for hemadsorptionindicating inactivation of more than 3 logs of virus; D, degeneration of leukocytes due to toxicity ofdisinfectant solution.

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TABLE 2. Effect of 0.1% solutions of variousdisinfectants on African swine fever virusa

Cultures withhemadsorption/cultures

Disinfectant inoculated

10-2° 10-'b 10-4"

Amphyl .................. 2/4 0/4 0/4Environ .................. 0/4 0/4 0/4Environ D ................ 0/4 0/4 0/4One-Stroke Environ ....... 0/4 0/4 0/4LpH ..................... 4/4 1/4 0/4

pHisoHex ................. 3/4 0/4 0/4SDS ................... 0/4 0/4 0/4Triton X-100 ............. 3/4 0/4 0/4Weladol .................. 2/4 0/4 0/4Distilled water control .... 4/4 4/4 4/4c

a 0.1% solutions of disinfectants were made indistilled water. Stock virus was diluted 10x indisinfectant solution and held at 22 to 25 C for 1 hr.Further serial 10-fold dilutions were made in culturemedium and titrated in leukocyte cultures. Stockvirus diluted 10x in distilled water and held for 1 hrwas similarly titrated.

bDilutions of virus-disinfectant mixture.c Stock virus in distilled water registered 4/4 out

through 10-7 dilution.

TABLE 3. Effect of some disinfectants on Africanswine fever virus determined by inoculation of pigs

First Day

DisinfectantaConcn Pig day of of

no. fever death

(DPI)b (DPI)

SDS.0.25 1' 6 11

1.0 2 6 10Environ D 0.25 3 6 9

1.0 4 7 10Environ 0.25 5 5 8

1.0 6 6 10One-Stroke Environ 0.25 7 5 9

1.0 8 12 15Virus control (10x dilu- 9" 5 8

tion of stock virus) 10 4 8

a The disinfectant solutions were made up in tapwater. Stock virus was diluted 10x in disinfectantsolution and held for 1 hr at 22 to 25 C. A pig was theninoculated intramuscularly with 1 ml of the mixture.

b DPI, day postinoculation. Note: An initial febrileresponse occurring 3 or more days after that of thefirst responding animal in the room is indicative of a

contact infection and cannot be attributed to theinoculum given.

c Pigs no. 1 through 8 were housed together.d Pigs 9 and 10 were housed in another room.

TABLE 4. Effect of concentration on inactivation ofAfrican swine fever virus with One-Stroke Environa

Disinfectant First day Day ofconcn

Pig no. of fever deathconcn(%) ~~~~(DPI)"' (DPI)

0.25 11 6 912 5 7

0.5 13 16 1814 9 13

0.75 15 15 1916 -c

1.0 17 16 1818 11 15

Virus control 19 3 720 3 8

a Stock virus was diluted 10x in the disinfectantsolution and held for 1 hr at 22 to 25 C. Each of twopigs was inoculated intramuscularly with 1 ml of thevirus-disinfectant mixture. Pigs 11 through 18 werehoused together. Pigs 19 and 20 were kept in aseparate room.

"DPI, day postinoculation. Note: An initial febrileresponse occurring 3 or more days after that of thefirst responding animal in the room is indicative of acontact infection and cannot be attributed to theinoculum given.

c Pig 16 did not have a fever and was still appar-ently normal at 30 DPI.

TABLE 5. Effect of time on inactivation of Africanswine fever virus with 1% One-Stroke Environa

First day Day ofInact(mtiotime Pig no." of fever death(mm) ~~~~~(DPI)c (DPI)

15 21 6 1022 12 14

30 23 8 1224 11 14

60 25 11 1426 11 14

120 27 13 1828 11 14

Virus control 29 4 830 4 7

a Stock virus was diluted 10x in 1% One-StrokeEnviron and held at 22 to 25 C. Samples taken at theindicated times were injected immediately (1 mlintramuscularly).

" Pigs 21 through 28 were housed together. Pigs 29and 30 were held in a separate room.

c DPI, day postinoculation. Note: An initial febrileresponse occurring 3 or more days after that of thefirst responding animal in the room is indicative of acontact infection and cannot be attributed to theinoculum given.

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TABLE 6. Ingredients of One-Stroke Environ (lIE)tested singly and in various combinations for their

effect on African swine fever virusa

Hemad-Concn in sorption

Compound 1% 1/E in leu-(mg/ml) kocyte

cultureb

o-Phenylphenol 1p-tert-Amylphenol 0.2 +o-Benzyl-p-chlorophenol 0.85 -

o-Phenylphenol plus p-tert-am- 1 + 0.2 -

ylphenolo-Phenylphenol plus o-benzyl- 1 + 0.85 -

p-chlorophenolo-Benzyl-p-chlorophenol plus p- 0.85 + 0.2 -

tert-amylphenolo-Phenylphenol plus p-tert-am- 1 + 0.2 -

ylphenol plus o-benzyl-p- + 0.85chlorophenol

a The solutions were prepared with distilled water,and in each instance the compound was in theconcentration it would be in a 1% solution of 1/E. Thestock virus (106 5 hemadsorbing units per ml) wasdiluted 10x in the test solution and held for 1 hr at 22to 25 C. The mixture was further diluted 100x inculture medium, and 1 ml was put on each of fourleukocyte cultures.

b +, Positive hemadsorption reaction, indicatingpresence of active virus; -, no reaction, indicatinginactivation of more than 3 logs of virus.

was recovered from its spleen.The other pig housed in the room did not

develop a fever or signs of ASF and was killedon the 10th day as ASF developing at this timecould be considered to originate from the firstpig.The four pigs housed in the previously ASF-

contaminated room that had been sprayed with1% 1/E did not develop ASF, nor were ASFprecipitating antibodies detectable in theirsera when the experiment was terminated after21 days.Table 6 shows the results obtained when the

stock virus was exposed to each of the activeingredients of l/E and assayed in LC. Therewas no sign of hemadsorption in o-phenyl-phenol, o-benzyl-p-chlorophenol, or in mix-tures containing these compounds, but hem-adsorption did occur in the p-tertiary-amyl-phenol-virus mixture. Toxicity of the com-pounds was tested at the same time, and,although some cytotoxicity was evident, therewas a sufficient number of intact leukocytesand red blood cells available to indicate thepresence of virus by hemadsorption. The re-sults of inoculating these compound-virusmixtures into pigs are shown in Table 7.Compared to the virus control pigs, there wasno difference in the first day of temperature (3days postinoculation [DPI ]) in the pigs inocu-

TABLE 7. Viricidal effect of the active components in One-Stroke Environ (lIE) on African swine fever virusas assayed in pigsa

First day Day ofCompound Concn in 1/E (mg/ml) Pig no. of fever death

(DPI)c (DPI)

o-Phenylphenol 1.0 31 6 1732 5 10

p-tert-Amylphenol 0.20 33 3 734 3 7

o-Benzyl-p-chlorophenol 0.85 35 3 1236 5 10

o-Phenylphenol + p-tert-Amylphenol + 1.0 + 0.20 + 0.85 37 6 15o-benzyl-p-chlorophenol 38 7 15

Virus controls 39 3 640 3 8

a The solutions were prepared with distilled water. The stock virus (106-5 hemadsorbing units per ml) wasdiluted 10x in the test solution and held for 1 hr at 22 to 25 C. The mixtures were further diluted 10x in cellculture medium, and two pigs were inoculated with each (1 ml intramuscularly).

t Pigs 31 through 38 were housed together. Pigs 39 and 40 were held in a separate room.c DPI, day postinoculation. Note: An initial febrile response occurring 3 or more days after that of the first

responding animal in the room is indicative of a contact infection and cannot be attributed to the inoculumgiven.

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lated with p-tertiary-amylphenol virus andessentially no difference in the day of deaththat was due to ASF virus. One of the two pigsinoculated with o-benzyl-p-chlorophenol andvirus developed a fever on the third day andwas dead 12 DPI. The other pig had a fever 5DPI and died 10 DPI. Both pigs inoculatedwith the o-phenylphenol-virus had tempera-tures at 5 and 6 days, respectively; one died 10DPI and the other on day 17. In contrast, pigsinoculated with the mixture of the activecomponents of 1/E did not develop a fever untilday 6 and 7 and both died on 15 DPI.There are several soluble antigens associated

with ASF virus (15) that may be readilydemonstrated by the agar gel diffusion precipi-tation reaction and immunoelectrophoresistests.

After 30 min of reaction with 1% 1/E, one ofthe soluble antigens was greatly diminished,and after 60 min it could no longer be detected(Fig. 1). Electrophoretically, this antigen at pH8.6 had very little migration in contrast to mostsoluble antigens that migrate toward the posi-tive electrode.

DISCUSSIONIdeally, each virus-inactivant mixture

should have been tested in pigs housed inseparate rooms. Because of space limitations,this was impossible. Therefore, it was neces-sary to depend upon previous experience con-

cerning contact transmission of ASF virus. TheLisbon 60 isolate was chosen for these experi-ments because it was involved in an extensiveoutbreak in domestic swine; it was known topersist and spread readily under field condi-tions; and its clinical manifestations were quiteuniform and well defined. The onset of feverwas a fairly reliable indicator of when infectionhad occurred. Since it was known that ASFvirus may appear in pharyngeal and nasalexcretions as early as 2 days before fever (4), itwas reasonable to assume that an animalhaving its first febrile response more than 2days after that of the first reactor in the roomwas either infected by contact with the firstinfected animal or had received an inoculumcontaining an extremely low level of activevirus.The compounds tested were representative of

the common classes of disinfectants and deter-gents, namely, halogen derivatives, substitutedphenols, polyphosphates, quaternary amines,nonionic and anionic surface-active com-pounds. All were evaluated by the same crite-rion, i.e., the effectiveness of a 1% solution as aninactivant of ASF virus suspended in a protein-rich menstruum. From the data in Tables 1 and2, it is evident that many commercially avail-able disinfectants have a marked degree ofviricidal activity against ASF virus. Amongthese are Amphyl, pHisoHex, Weladol, andTriton X-100, besides those tested in pigs

FIG. 1. Effect of One-Stroke Environ (lIE) on the soluble antigens of African swine fever virus. Untreatedsoluble antigens are on the left, and the lIE-soluble antigen mixtures are on the right on each slide. A currentof 2 ma/cm was applied for 90 min, and the precipitin patterns were developed with ASF survivor pig serum.One fast moving antigen was apparently destroyed immediately. The progressive disappearance of the antigenclosest to the negative electrode during the 60-min treatment is evident.

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(Table 3). Under conditions of higher concen-trations, longer time of inactivation, or a men-struum having a lower protein content, theymay well be effective inactivants for ASF virus.

It appears from Tables 6 and 7 that the mosteffective viricidal ingredient against ASF virusin One-Stroke Environ is o-phenylphenol. Thiscompound is a common ingredient in manycommercially available disinfectants and iseffective in destroying hog cholera virus (18;H. S. Wright, Bacteriol. Proc., p. 10, 1971).Its use for that purpose has been approvedby the Animal and Plant Health Division ofthe U.S. Department of Agriculture (12). The1% solution of o-phenylphenol recommended asa disinfectant for hog cholera is 10 times theconcentration used in our experiments (Tables6 and 7) and should likewise serve to inactivateASF virus under field conditions.

Since ASF virus is sensitive to ether andchloroform and its nucleic acid is readily ex-tractable by both phenol and SDS (1), it isprobable that the mechanism of its inactiva-tion by phenolic derivatives is solubilization ofthe lipid segments of the virus envelope. Whenthe antigenic components of the virus havebeen characterized, the observation made withimmunoelectrophoresis will be more meaning-ful, and may perhaps be indicative of themechanism of inactivation.

Since certain Argasid ticks are capable ofharboring and transmitting ASF virus for manymonths (8), disinfection of a previously in-fected premise may also require elimination ofthese arthropods before restocking is consid-ered.

ACKNOWLEDGMENTSThe interest and encouragement of J. Hyde and A. H.

Dardiri and technical assistance of M. Llewellyn and L.Taplin are greatly appreciated.

LITERATURE CITED1. AdIdinger, H. K., S. S. Stone, W. R. Hess, and H. L.

Bachrach. 1966. Extraction of infectious deoxyribo-

nucleic acid from African swine fever virus. Virology30:750-752.

2. Callis, J. J., and G. E. Cottral. 1968. Methods forcontainment of animal pathogens at the Plum IslandAnimal Disease Laboratory, p. 465-480. In K. Mara-morash and H. Koprowski (ed.), Methods in virology,vol. 4. Academic Press Inc., New York.

3. Fellows, 0. N. 1960. Chemical inactivation of foot-and-mouth disease virus. Ann. N.Y. Acad. Sci. 83:595-608.

4. Grieg, A. S., and W. Plowright. 1970. Excretion of twovirulent strains of African swine fever virus by do-mestic pigs. J. Hyg. 68:673-682.

5. Hess, W. R. 1971. African swine fever virus, p. 1-33. In S.Gard, C. Hallauer, and K. F. Meyer (ed.), Virologymonographs, vol. 9. Springer-Verlag, New York.

6. Hess. W. R., B. F. Cox, W. P. Heuschele, and S. S.Stone. 1965. Propagation and modification of Africanswine fever virus in cell cultures. Amer. J. Vet. Res.26:141-146.

7. Hess, W. R., and D. E. De Tray. 1960. The use ofleukocyte cultures for diagnosing African swine fever.Bull. Epizoot. Dis. Afr. 8:317-320.

8. Heuschele, W. P., and L. Coggins. 1965. Studies on thetransmission of African swine fever virus by arthro-pods. Proc. U.S. Livestock Sanit. Ass. 69:94-100.

9. Kovalenko, J. R., M. A. Sidarov, and L. G. Burba. 1965.Experimental investigations on African swine fever.Bull. Off. Int. Epizoot. 63:169-189.

10. Malmquist, W. A., and D. Hay. 1960. Hemadsorptionand sytopathic effect produced by African swine fevervirus in bone marrow and buffy coat cultures. Amer. J.Vet. Res. 21:104-108.

11. Manso Ribeiro, J., and J. A. Rose Azevedo. 1961. Lapeste porcine africaine au Portugal. Bull. Off. Int.Epizoot. 55:88-106.

12. Mulhern, F. J. 1970. Hog cholera and other communica-ble hog diseases. Title 9, Part 76, Oct. 29, 1970. InFederal Register 35, 16731.

13. Reporte preliminar del brote de fiebre povcina Africanaen Cuba. 1971. Instituto Nacional de Medicina Veteri-navia, Habana.

14. Scheidegger, J. J. 1955. Une micro-methode de l'im-mune-electrophorese. Int. Arch. Allergy 7:103.

15. Stone, S. S., and W. R. Hess. 1965. Separation of virusand soluble noninfectious antigens in African swinefever by isoelectric precipitation. Virology 26:622-629.

16. Stone, S. S., and W. R. Hess. 1967. Antibody response toinactivated preparations of African swine fever inpigs. Amer. J. Vet. Res. 28:475-481.

17. Sykes, G. 1965. Disinfection and sterilization. J. B.Lippincott Co., Philadelphia.

18. Torrey, J. P., and W. C. Amtower. 1964. Inactivation ofhog cholera virus in blood and excreta with chemicaldisinfectants. Proc. U.S. Livestock Sanit. Assoc.68:287-297.

122 APPL. MICROBIOL.

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ERRATA

Effects of Some Disinfectants on African SwineFever Virus

S. S. STONE AND W. R. HESSPlum Island Animal Disease Laboratory, Greenport, Long Island, New York 11944

Volume 25, no. 1, p. 118, Table 1, column 2, last two lines: Change "3% Acetic acid" to "2%Acetic acid" and change "2% Acetic acid" to "2% Sodium hydroxide."

Itroduction of Pseudomonas aeruginosa intoa Hospital via Vegetables

SPYROS D. KOMINOS, CHARLES E. COPELAND, BARBARA GROSIAK, AND BOSKO POSTICDepartments of Pathology and General Surgery, Mercy Hospital, Pittsburgh, Pennsylvania 15219, and

Department of Epidemiology and Microbiology, Graduate School of Public Health, University ofPittsburgh, Pittsburgh, Pennsylvania 15213

Volume 24, no. 4, p. 567, column 2, paragraph 4, line 2: Change "0.3% cetrimide" to "0.03%cetrimide."