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Indirect hemagglutination test that uses glutaraldehyde fixed sheep erythrocytes sensitized with extract antigens for detection of Pasteurella antibody

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0095-1137/82/050752-05$02.00/0

Indirect Hemagglutination

Test

That Uses

Glutaraldehyde-Fixed Sheep Erythrocytes Sensitized with

Extract

Antigens

for Detection of Pasteurella

Antibody

TAKUO SAWADA,* RICHARD B. RIMLER,ANDKEITH R. RHOADES

National Animal Disease Center, Agricultural Research, Science and EducationAdministration, U.S.

Department

of Agriculture,

Ames,Iowa50010

Received 23 July1981/Accepted 29 December 1981

Glutaraldehyde-fixed sheep erythrocytes (GA-SRBC)wereused in the indirect

hemagglutination testfor the detection of Pasteurella antibody. GA-SRBCwere

stable for at least 6 months. Heat extract or potassium thiocyanate extract

antigens of Pasteurella strains could be adsorbed ontoGA-SRBCortanned

GA-SRBC, respectively. The indirect hemagglutination test reaction was capsular

group specific with heat extractantigen-sensitized GA-SRBC but not potassium

thiocyanate extract antigen-sensitized tanned GA-SRBC.

Indirect

hemagglutination

(IHA) tests have

been employed

successfully for

the

identifica-tion of

serological

types

of Pasteurella

multo-cida

(4) and P.

haemolytica

(1). In

addition,

Carter and Rappay

(5)

have

described

a

modified

IHA test

that

uses

formalinized

group 0 human

erythrocytes (RBC).

Although

group 0 human

RBC

have

been

found to be the most

satisfac-tory

erythrocytes

for

the

serotyping of

P. multo-cida(4)

strains, they

are

difficult for

some

labo-ratories to obtain.

Also,

formalinized,

sensitized

RBC have a tendency to clump and thus become

unsuitable

for

the assay

of

antibody

(6).

Na-mioka

and Murata (10) have

demonstrated

that

sheep RBC (SRBC)

canbe

substituted for

group

O human

RBC. Mukker

and

Nilakantan

(8, 9)

have

described

an IHA test that uses tannic

acid-treated SRBC

to detect

antibody

in cattle

immunized with

P.

multocida.

Noattemptto use

SRBC fixed

with agents other than

Formalin

in

the IHA test to

detect Pasteurella

antibody

has

been

reported.

The purpose

of

this report is to describe a

modified IHA test that uses

glutaraldehyde-fixed SRBC

(GA-SRBC)

for the detection of

Pasteurella

antibody.

MATERIALSANDMETHODS

Bacteria. Fourstrains of P. multocida andonestrain

ofP. haemolytica were used. Strains 1059 and P-3827ofP. multocida,isolatedfromaturkeythatdied offowlcholera andfromabovinethat hadpneumonia, respectively, areclassified inCarter'scapsulargroup A. Strains P-1256 and P-1235 of P.multocida,

respec-tively isolated fromabuffalo andabovinethatdied of hemorrhagicsepticemia, areincapsulargroups B and

E,respectively. Strain L-101 ofP. haemolytica

sero-type 1wasisolated from thelungofacalf that diedof

acuterespiratorydisease. Colonies of all strains grown

on dextrosestarch agar (DSA) plateswereiridescent in obliquetransmittedlight, indicating that the bacteria

wereencapsulated. P.multocidaP-3827alsoproduced

mucoid colonies.

Rabbit antisera. We prepared anti-P. multocida

group A serumby immunizingyoungadult maleNew

Zealand whiterabbits with Formalin-killed P.

multo-cidaP-1059cells. The rabbitswereinoculated subcu-taneouslywith 0.1 mlofacellsuspension (equivalent indensityto aMcFarlandnephelometer standardof1)

preparedfroma4-h culturegrown onDSA.

Thereaf-ter,therabbitswereinoculated intravenouslyat

week-ly intervals for5weeks:weadministered0.2, 0.3, 0.4,

0.5, and0.6mlatweeks1,2,3, 4,and 5,respectively. Serawerecollected from the rabbits1 week after the lastinjection and preserved with 0.01% thimerosaland

0.06%phenol at4°C. Anti-P. mlultocida groupB and

groupE sera, described previously(12), were used.

Anti-P. haemolytica serotype 1 and serotype 2 sera were kindly providedby G. H. Frank, National Ani-mal Disease Center. The sera were prepared as de-scribedby Frank and Wessman(7).

Heat extractantigen. The cells of P. multocida

P-3827, P-1256 and P-1235 and P. haemolytica L-101,

grown onheavily seeded DSA in standard petri plates

for 18 h at 37°C, were harvested in 1 ml of 0.02 M

phosphate-bufferedsaline(PBS; pH 7.2)perplate.The cellsuspensionofP.multocida P-3827 was mixed with

anequalvolumeof PBScontaining200 Uof

hyaluroni-dase (bovine testis; Calbiochem) per ml, and this mixture was incubatedat37°C for2h withoccasional

shaking to remove hyaluronic acid, which interferes with the IHA test. All of the cell suspensions were heated at 100°C for 1 h. The cells were pelleted by centrifugation at 10,000 x g for 20 min, and the supernatantfluidwasdesignatedheatextractantigen.

The extractantigenswerepreservedwith0.1%sodium

azideandkeptat4°C.

KSCNextractantigen.Cells harvested in PBS from

DSA culturesasdescribed abovewerewashed twice with PBSbycentrifugation.The washedcells,except

for P. multocida P-3827cells, weresuspended in 0.5

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ml of PBS per plate and mixed withanequal volume of PBS containing 2.0 M potassium thiocyanate (KSCN) (pH 7.0). BecauseP.multocida P-3827 cells couldnot

beeasily suspended inonly 0.5mlofPBS, theywere

suspended in 1.5 ml and then treated in the same

manner as the other cell suspensions. Each mixture was stirred at4°C for 2 h. The cellswerepelleted by centrifugationat10,000x gfor20min (orat14,500 x

g for 40 min forP.multocidaP-3827), and the superna-tant fluid was removed by aspiration. The pelleted cellsofeachplate were suspendedin 1.0 ml of PBS containing1.0 MKSCN(pH 7.0) and stirredat4°C for

2 h. The cells were pelleted by centrifugation as

described above, and the supernatant fluids from the first and second extractions were combined. The

ex-tractedantigenswerefilteredthrougha0.45-,um

mem-branefilter(MilliporeCorp.) and dialyzed exhaustive-ly against PBS. The antigens were preserved with 0.1% sodium azide andkeptat4°C.

Fixation of SRBC. SRBC were treated with glutar-aldehyde byamodification of the method ofBingetal. (3). A 100-ml suspension of fresh SRBC in Alsever solutionwaswashedby centrifugation (650x gfor20

min) six times with fiveorsix volumes of saline(0.85%

NaCl). After the last wash, the packed cells were

suspended in PBStoyielda10%suspension (vol/vol)

and chilledto4°C in an ice bath. A 25% solution of glutaraldehyde (Eastman Kodak Co.)was diluted to

1% (vol/vol) with PBS and chilled to 4°C. A 10% suspension of washed SRBCwasmixed withanequal

volume of the1% solution ofglutaraldehyde, and the mixture was incubatedat 4°Cfor 30 min withgentle

stirring.The mixturewasthencentrifugedat650 x g

for 10 min at 25°C. The pelleted, fixed cells were

suspended inPBS, washed three times with PBS by

centrifugation,andsuspendedinPBScontaining0.1%

sodium azide to yield a 10% suspension. The GA-SRBCwerestoredat4°C.

T-GA-SRBC. The GA-SRBC tobe sensitizedwith

protein antigens of the KSCN extract were treated withtannic acid.A10% suspension of GA-SRBC was mixed withanequal volume of PBS containing 0.005% tannic acid(wt/vol), and the mixture was incubated at 37°C for30min with occasional shaking. The tanned GA-SRBC(T-GA-SRBC)werepelleted by centrifuga-tion at650 x gfor10 minat 25°C and washed three times with PBS. After the last wash, the T-GA-SRBC

weresuspended in PBS to yielda10% suspension. SensitizationofGA-SRBCwith heat extractantigen.

A 10% suspension of GA-SRBC was mixed with an equal volume of a serial twofold dilution of heat

extractantigen. The mixturewasincubatedat37°C for

1hwith occasionalshaking. The sensitized cells were washed three times with PBS by centrifugation and suspended in PBS containing 0.25% bovine serum

albumin(BSA-PBS)and0.1%sodium azide to yield a 0.5% suspension (vol/vol). The optimal dilution of antigen forthe sensitization of GA-SRBC was deter-mined by box titration against homologous rabbit

antiserum. Thedilution of antigen used in the IHA test was the fourfold-higher (4 hemagglutination units)

concentration thatshowed thehighest specific titer in

theantiserum (1 hemagglutination unit). Fresh SRBC washed three times with PBS were sensitized with heatextractantigen ofP.multocida P-1256 or P-1235,

and abox titration was done as described above for comparisonwithsensitizedGA-SRBC.

SensitizationofT-GA-SRBCwithKSCNextract anti-gen. A 10% suspension of T-GA-SRBC was mixed with an equal volume ofa serial twofold dilution of KSCN extractantigen. The mixturewasincubatedat

37°C for 30 min withoccasional shaking. The sensi-tized cellswerewashed threetimeswithBSA-PBSby centrifugation andsuspended in BSA-PBS to yielda

0.5% suspension (vol/vol). The optimal dilution of antigen for the sensitization of T-GA-SRBCwas deter-minedby box titration.

IHA test. The IHA test was performed with a

microtiter system (DynatechLaboratories, Inc.). Seri-altwofold dilutions of antiserumwere made in BSA-PBS, and 0.025 mlof the sensitized SRBCwasadded

to 0.025 ml of the antiserum dilution in U-bottom plates. The plates were shaken and allowed to stand for1 to 2hat25°C before SRBC settling patternswere

read. TheIHAtiterwasexpressedasthereciprocal of the highestdilution of serumshowing adefinite posi-tive pattern (flat sediment), as compared with the pattern of the negative control (smooth dot in the centerof the well). Controls consisted of unsensitized SRBC plus test serum and sensitized SRBC plus diluent. Whenheterophile antibodiesweredetected in sera,they were removed by absorption with unsensi-tized GA-SRBCat25°C for2hbeforebeing used inthe IHAtest.

RESULTS

Antigen titration.

Initial experiments

con-firmed

that a heat extract

antigen of

P.

multo-cida

P-1256 or P-1235 was

adsorbed

onto

fresh

SRBC and that the sensitized SRBC

were

specif-ically agglutinated

by

rabbit

antiserum. The

GA-SRBC sensitized with

heatextract

antigen of

P.

multocida

P-1256 or P-1235 showed the same

results

in box titration

as were shown by

fresh

SRBC. Typical results of a box titration of heat

extract

antigen-sensitized GA-SRBC

are shown

in Table 1. An excess

of antigen (dilutions of

1:16 and

below)

slightly inhibited

IHA (P.

mul-tocida

P-1235

and

P.

haemolytica

L-101

[Table

1]). Almost identical titers

were

obtained with

the same

antisera when several different lots of

sensitized GA-SRBC

were

prepared and tested

during the

course

of

the experiment. The

GA-SRBC

were

stable

for

at least 7 months and

adsorbed the

heat extract antigens of

Pasteur-ella

strains. Controls of unsensitized GA-SRBC

in each

dilution

of the antiserum and controls of

GA-SRBC sensitized with antigen

in

diluent

showed definite negative patterns.

Typical

results of a box titration of KSCN

extract

antigen-sensitized

T-GA-SRBC are

shown in Table 2. The antibody titer of rabbit anti-P. multocida group A serum was low

against

the KSCN extract antigen of P.

multo-cidaP-3827, and this result differed from that for

the

antigen of

other Pasteurella strains. Identi-cal

antibody

titers were obtained with the same

serawhen several different lots of sensitized

T-GA-SRBC

were prepared and tested. For at 5,

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TABLE 1. Typical results of box titration of

GA-SRBC sensitized withheatextract antigens and

homologous rabbitantisera

IHAtiter inantiserum againsttheantigen ofa

Antigen P.multocida P.

dilution haemolytica

P-3827(A)b P-1256 (B) P-1235 (E) L-101 (1)

1:1 1,024 NDc ND 512

1:2 1,024 ND ND 512

1:4 1,024 ND ND 1,024

1:8 512 1,024 2,048 1,024

1:16 256 1,024 2,048 2,048

1:32 64 1,024 4,096 4,096

1:64 <4 1,024 4,096 4,096

1:128 <4 512 4,096 4,096

1:256 <4 <4 4,096 4,096

1:512 <4 <4 <4 1,024

1:1,024 <4 <4 <4 64

aTitersareexpressedasthereciprocals oftheinitial

dilutions ofserum.

bLetter inparentheses indicates the capsulargroup;

number indicates theserotype.

c ND, Notdone.

TABLE 2. Typicalresults ofbox titration of

T-GA-SRBC sensitized with KSCNextractantigenand

homologous rabbit antisera

IHAtiter in antiserumagainst theantigenofa:

Antigen P.multocida P.

dilution haemolytica

P-3827(A)b P-1256(B) P-1235 (E) L-101(1)

1:1 32 1,024 4,096 2,048

1:2 32 1,024 4,096 2,048

1:4 32 1,024 4,096 2,048

1:8 32 1,024 4,096 2,048

1:16 16 1,024 4,096 1,024

1:32 8 512 4,096 64

1:64 <4 256 <4 64

1:128 <4 <4 <4 32

1:256 <4 <4 <4 <4

aTitersareexpressedasthereciprocalsoftheinitial

dilutions ofserum.

bLetter inparenthesesindicatesthecapsulargroup;

number indicates the serotype.

least 6 months duringthe courseof the

experi-ment,GA-SRBCwereabletoadsorb the KSCN

extractantigen of Pasteurella strains satisfacto-rily after the treatment with tannic acid. Con-trols in the IHA test always showed definite

negativepatterns.Antigen-sensitized GA-SRBC

orT-GA-SRBC could be used foratleast 1week inthe IHAtestwithoutanylossof

agglutinabili-tyby homologous rabbitantiserum.

Specificityof the IHAreaction. IHAcrosstests

were performed to determine the specificity of

thereaction in the IHAtestwithheatorKSCN

extract antigen. Capsular group-specific

reac-TABLE 3. IHA cross testof heatextract

antigen-sensitizedGA-SRBC with rabbit antiserum

IHA titer inantiserumofb:

Antigena P.multocida P.

G haemolytica

GroupA GroupB Group E serotype1

P-3827(A)c 2,048 <4 <4 <4 P-1256(B) <4 2,048 <4 <4 P-1235(E) <4 <4 8,192 <4

L-101(1) <4 <4 <4 8,192

aTheoptimal dilution of each antigenwasused for

the sensitization ofGA-SRBC.

bTitersareexpressed as the reciprocals ofthe final dilutions ofserum.

cLetter inparentheses indicates the capsular group;

number indicates the serotype.

TABLE 4. IHA crosstest of KSCNextract antigen-sensitizedT-GA-SRBC with rabbitantiserum

IHA titer in antiserumofb:

Antigena P.multocida P.

haemolytica

GroupA GroupB Group E serotype1

P-3827(A)c 64 <4 <4 <4 P-1256(B) 256 2,048 64 8,192 P-1235(E) 32 64 8,192 1,024 L-101 (1) 512 256 512 4,096

aTheoptimal dilution of each antigen was used for thesensitization ofT-GA-SRBC.

bTitersareexpressed asthe reciprocalsofthe final dilutions ofserum.

cLetter in parentheses indicates the capsular group; numberindicates the serotype.

tions were demonstrated for the Pasteurella

strains(Table 3).GA-SRBC sensitized withheat extract antigen ofP. haemolytica L-101

(sero-type 1) did not react with rabbit anti-type 2 serum(datanotshown).

The results of IHA cross tests of KSCN extract antigen-sensitized T-GA-SRBC are shown in Table 4. Capsulargroup-specific

reac-tions in IHA tests were not seen, with the

exception ofP. multocida P-3827. The KSCN

extract antigen ofP. multocida P-3827 showed

negative

reactions

against

heterologous

antise-rumandalowpositivereactionagainst

homolo-gous antiserum. The IHA titers for all strains

weregenerally

higher

in

homologous

reactions

thanin

heterologous

reactions.

When GA-SRBC that had not been treated

with tannic acid were sensitized with KSCN

extractantigenof either P. multocida P-1256 or

P-1235, an IHA titer of1,024 or 2,048,

respec-tively,

wasobtained

against

homologous

antise-rum. No reactions occurred with heterologous

antisera.

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IHA TEST WITH GA-SRBC 755

DISCUSSION

The

GA-SRBC

can be used for at least 7

months when stored at

4°C.

This saves time that

wouldbe spent in preparing fresh SRBC

suspen-sions

and increases

the

reproducibility of

the

test because the same RBC lot

is

used.

There-fore,

the useof

GA-SRBC

in the IHAtestshould

beuseful for the

serological

study of

pasteurello-sis.

Carter and Rappay(5) have described a

modi-fied IHA test that uses formalinized group 0

human RBC.

Although

SRBC were not

com-pared

with

human RBC in our

experiment,

SRBC

always showed either distinct

hemagglu-tinating

or

non-hemagglutinating

patterns when-ever

fixed

or

fresh

cells were used. We believe that

SRBC

can be

substituted

completely

for

group 0

human

RBC.

Lysis

of bovine

RBC at low serum dilutions

has been observed in the IHA test

for

the

serotyping of

P.

haemolytica strains (1).

Recent-ly, Frank and Wessman (7) reported that bovine

RBC sensitized with the P.

haemolytica

type 12

isolate

are

hemolyzed in

theIHA

reaction,

mak-ing

interpretation difficult. Frank

and Wessman

noted

that

hemolysis

occurs even when the

antiserum has

been

heat

inactivated

and

ad-sorbed with bovine RBC.

If

fixed SRBC

are

employed in

the IHA test

for the

serotyping

of P.

haemolytica

strains, hemolysis

can

possibly

be

eliminated.

Studies have shown that

formalinized

cells

tend to

clump after

storage

for

6 months to 1

year at

-20°C,

thereby becoming

unsuitable

for

antibody

assays. In

addition,

uniform

suspen-sions of formalinized

cells are

difficult

to pro-duce

(3, 6).

GA-SRBC

prepared

and stored at

4°C

in the present

experiment

consistently

and

easily produced

a

uniform

cell

suspension

when

stirred,

and

unsensitized GA-SRBC

or

T-GA-SRBC

always produced definite negative

pat-terns

(smooth dot

in the center

of

the

well)

in

antiserum

or

diluent.

Heat extract

antigen-sensitized GA-SRBC

re-acted

only

with

homologous

antiserum,

confirm-ing previous

results

obtained

when sensitized

group 0 human RBCwere used (12). Therefore,

webelieve that an IHA test that uses GA-SRBC

would be useful for the detection of capsular

group-specific

P. multocida antibody. The type

specificity

of the IHA reaction with the heat

extract

antigen

of P. haemolytica L-101 was

determined only against

anti-serotype

1 and

se-rotype2sera. Ourfindings suggest that this test

would be useful for the detection of the

type-specific antibody against

P.

haemolytica

sero-type 1 strains in cattle: P. haemolytica serotype

1 strainspredominate in cattle (1, 2, 7).

The KSCN extract

antigen-sensitized

T-GA-SRBC

reactedwith both homologous and

heter-ologous group antisera. The negative reaction of

P. multocida P-3827

antigen

with

heterologous

antiserum was considered tobe due tothe low

antigenicity of the strain, as determined by the

low IHA titer of the homologous antiserum.

The IHA test was capsular group-specific

whenantigens in the KSCN extract of P.

multo-cida P-1256 or P-1235 were adsorbed onto

GA-SRBC but not T-GA-SRBC. This suggests that

protein antigens, common to P. multocida

P-1256 and P-1235, in the KSCN extracts were

adsorbed only onto T-GA-SRBC.

In this study, the results of IHA tests that

usedT-GA-SRBC sensitized with KSCN extract

antigens indicated that cross-reactions between P.

multocida and

P.

haemolytica took

place.

Similar cross-reactions

have

been

reported by

Mukkur (8), who used tanned SRBC sensitized

with P. haemolytica KSCN extract

antigens

to

demonstrate antibody in calves inoculated with

P. multocida KSCN extract. These

cross-reac-tions suggest that there areantigens common to

these pasteurellae that are extractable with

KSCN.

Antigens

common to these

pasteurellae

have beendemonstrated

previously by

immuno-diffusion methods

(11). Common

antigens

ex-tracted

with

KSCN

and used to

sensitize

T-GA-SRBC can

perhaps

be used in a

single

IHA test to

demonstrate antibody against either

P.

multo-cida

orP.

haemolytica.

ACKNOWLEDGMENT

We thank William E. Brown and Lance L. Ferguson for technicalassistance.

LITERATURECITED

1. Biberstein, E. L., M. Gills, and H. Knight. 1960. Serologi-cal typesof Pasteurellahemolytica.Cornell Vet. 50:283-300.

2. Biberstein, E.L.,and D. A.Thompson. 1966. Epidemio-logical studieson Pasteurella haemolytica in sheep. J. Comp.Pathol. 76:83-94.

3. Bing,D.H.,J. G.M.Weyand,and A.B.Stavitsky.1967. Hemagglutination with aldehyde-fixed erythrocytes for assayofantigens and antibodies. Proc. Soc. Exp. Biol. Med.124:1166-1170.

4. Carter,G. R. 1955.StudiesonPasteurella multocida.1. A hemagglutination testfor theidentification of serological types. Am. J. Vet. Res. 16:481-484.

5. Carter, G.R., and D. E. Rappay. 1962. Formalinized erythrocytes in the haemagglutination test for typing Pasteurella multocida. Brit.Vet. J. 118:289-292. 6.Daniel, T. M.1965. Observationon the antibodyresponse

of rabbits tomycobacterialantigens.J. Immunol. 95:100-108.

7. Frank, G. H., and G. E. Wessman. 1978. Rapid plate agglutination procedure for serotypingPasteurella hae-molytica.J.Clin. Microbiol. 7:142-145.

8. Mukkur, T. K. S. 1978. Immunologic and physiologic responseofcalvesinoculatedwithpotassiumthiocyanate extractof Pasteurella multocidatype A. Am. J. Vet. Res. 39:1269-1273.

9. Mukkur, T. K. S., and P. R.Nilakantan. 1969.The rela-tionshipofhemagglutinating antibody with protectionin cattleimmunized againsthemorrhagic septicemia.Cornell Vet.59:643-648.

10. Namioka, S., and M. Murata. 1964.Serologicalstudies on

VOL. 15, 1982

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Pasteurellamultocida. V.Some epizootiological findings resulting from0antigenic analysis. CornellVet. 54:520-534.

11. Prince,G.H.,and J. E. Smith.1969.Antigenicstudieson Pasteurellamultocidausingimmunodiffusion techniques.

II. Relationships with other Gram-negative species. J. Comp. Pathol. 76:315-320.

12. Rimler,R. B.1978. Coagglutination test foridentification of Pasteurella multocida associated with hemorrhagic septicemia.J. Clin. Microbiol. 7:142-145.

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