antibody response in rabbits infected with rochalimaea henselae, rochalimaea quintana and afipia...

A P M I S 102. 943-949. I994 Prinrrd in Dmniurh , All rights reserwd

C o p j r i g h t 0 A P M l S I994

/bJJ&dg ISSN 0903-4641

Antibodv response in rabbits infected with Rochalimaea henselae, Rochalimaea quintana

and Afipa felis

KRRSTEN ENGBRK and CLAUS KOCH

Department of Immunology, Section of Applied Immunology, Statens Seruminstitut, Copenhagen, Denmark

Engbrek, K. & Koch. C. Antibody response in rabbits infected with Rochulimuc~u henselue, Rochcili- inuea quintuna and A$piu/&lis. APMIS 102: 943-949, 1994.

Antibody responses in three pairs of rabbits inoculated with live Rochuliniue~i henselae, Rochaliinueci quintuna and AJipia,felis were studied by enzyme immunoassay with whole-cell and bacterial sonicates as antigen. No differences in measured antibody responses were found with the two types of antigen preparation. Two rabbits did not respond with antibody production. In the remaining rabbits there was a low-titred antibody response that showed no significant cross-reaction with related bacteria. After rechallenge the antibody response rose significantly and there was significant cross-reaction with related bacteria. The antigens involved in the antibody response were examined by crossed immunoelectrophoresis. After the initial inoculation 5-7 precipitin lines of the reference diagrams were deflected, including lines which cross-reacted with antigens found in related bacterial species. After reinoculation several more precipitin lines were deflected, including additional lines cross-reacting with antigens present in related bacteria and common bacterial antigens.

Key words: Rochulimueu henselue; Rochuliniueu yuintunu; AJipiu felis; infection model; enzyme immunoassay; immunoelectrophoresis.

K. Engblek, Department of Immunology, Section of Applied Immunology, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.

Several serodiagnostic tests have been assessed in the diagnosis of Rochulimueu infections. Sera from patients with trench fever have been in- vestigated by complement fixation (CF) ( lo) , passive haemagglutination (PHA) (4), radioimmunoprecipitation (6), enzyme immunoassay (EIA) and counterimmunoelectrophoresis (CIE) (7). The titres in CF and PHA tests were generally low and antibodies could not be demonstrated in all patients. All sera were positive in EIA and CIE, giving one to three precipitin lines in the latter. False-positive reactions were

Received August 15, 1994. Accepted October 27, 1994.

seen with sera from patients with Q fever and the typhus, spotted fever and scrub typhus group of rickettsiae. IgG appeared to be the major reactive antibody class.

Sera from suspected cat-scratch disease (CSD) have been tested for antibody to Ruchuli- maea henselae by the indirect fluorescence antibody (IFA) test (8) and EIA (1). By these two methods 88% and 95%, respectively, of the sera showed an elevated antibody titre, whereas 94% and 99% of healthy control sera were negative (titre <64). Ten sera had antibodies to Ajipiu felis in low titre. Except for a low IFA titre in 2 of 10 patients with brucellosis, no false-positive reactions were found in sera positive for Cuxiel- Iu hurnetii, Rickettsiu typhi, Rickettsia rickettsii,

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ENGBEK & KOCH

Yersinia pestis, Francisella tularensis, Trepone- ma pallidum, Borrelia burgdorferi, Mycoplasma pneumoniae, Chlamydia trachomatis, rheuma- toid factors, or alpha-fetoprotein.

In a previous investigation we analysed water- soluble extracts of R. henselae, R. quintana and A . felis by means of crossed immunoelectrophoresis, and demonstrated 56, 49 and 39 antigens in the respective extracts. Several antigens of R. henselae and R. guintana cross-reacted with antisera against the other species, whereas more than 30 antigens distinguished AJipia felis from the two Rochalimaea species (5 ) .

In the present investigation we have examined the antibody responses in rabbits inoculated with live R. henselae, R. quintana or A. felis, the proportion of cross-reacting antibodies mounted against related bacteria, and the ident- ity of the antigens against which antibodies are formed.

MATERIALS AND METHODS

Bacterial strains Rochaliniueu henselae ATCC 49882, Rochuliniueu

quintana ATCC VR 358 and AJipia felis ATCC 53690 were obtained from the American Type Culture Col- lection, Rockville, and Brucella ubortus biotype 1 ATCC 23448 came from the State Veterinary Serum Laboratory, Denmark.

Antigen preparation Rochalimaeu and Brucella species were grown on

blood agar base no. 2 (Difco 0696) with 5'%1 horse blood and AJipia ,felis on buffered charcoal-yeast ex- tract agar (Statens Seruminstitut product no. 262104). For the whole-cell antigen preparation the growth was scraped off the plates and washed three times in saline. After the final centrifugation the pel- let was resuspended in 50 mM sodium carbonate buffer, pH 9.6, and the turbidity was adjusted to OD650: 1.5. The Triton-soluble antigens were prepared by the sonication procedure described in (5). The protein

Development of antibodies in rabbits determined by EIA

Rahhir 2902h' hcnrclor

. " . " . . O R abhil 29031R hensrlae

.... o... R ahhil 2W4IR. q u i n r m

---I _--- Rahhil 2905lR. quinram

- - +- - - Rahhit 2906lA. Jclis

R vhhil 2907lA Jdds

. . 1 2 ; 4 5 6 1 8 9

Week

0 1 2 3 4 5 6 7 8 9 Week I

Development of cross-reacting antibodies in rabbit 2902 inoculated with R. henrelae.

+ assayed against R. henselae

assayed againsf R. q u i n m

-.--Q--- assayed againsf A. felis

---+---- asayed againsf 8. &nus

Fig. 1. Development of antibodies in rabbits inoculated with R. henselae, R. quintunu and A. jelis starting with the preinoculation level and tested weekly thereafter. Six weeks after the first inoculation the rabbits were rechallenged with the same organism and dose. The antibody level is given as ODAg0 ~ OD65O produced by sera diluted 1:800 and tested in microtitre plates coated with sonicated antigen (10 pgiml) of the immunizing bacteria.

Fig. 2. Development of cross-reacting antibodies to R. quintana, A . felis and Brucellu abortus in rabbit 2902 inoculated with R. henselae determined as ODA9" - ODh50 in EIA by sera diluted 1:800 and tested in microtitre plates coated with sonicated antigens (10 pgiml).

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R O C H A L I M A E A ,ND AFIPIA INFECTIONS IN RABBITS

Fig. 3. Development of cross-reacting antibodies to R. henselue, A. felis and Brucellu abortus in rabbit 2904 inoculated with R. quintana determined as OD490 ~ ODhso in EIA by sera diluted 1:SOO and tested in microtitre plates coated with sonicated antigens (10 pgiml).

Fig. 4. Development of cross-reacting antibodies to R. henselue, R. yuintunu and B. abortus in rabbit 2907 inoculated with A. felis determined as OD4y0 - OD,So in EIA by sera diluted 1:SOO and tested in microtitre plates coated with sonicated antigens (10 pgiml).

Xvelopment of cross-reacting mtihodies in rahhit 2904 inoculated Nith R. quintana.

+ awyed against R y r ! m n o

. - - - O assayed against R. henrehe

.... o...

--*-- assayed against B. &onus

assayed against A. fells

0 ; 2 ; 4 5 6 7 8 9

Wcrh

0 1 2 3 4 S h 7 1 We& I

Development of cross-reacting antibodies in rabbit 2907 inoculated with A. felis

-D- assayed against A. felts

........ 0

.._. 0 . . .

---*--- a%ayed against B. abortus

%\bayed against R. henseloe

avsayed against R. quintam

concentration was determined by the dye-binding as- say described by Brudford with BSA as the standard (3).

Sera Live R. henselue, R. quintunu and A. f d i s suspen-

sions at a concentration equal to McFarland turbidity standard No. 3 ( I S X 10' CFU/ml) were in- jected intracutaneously at a dose of 0.2 ml into three pairs of female rabbits, each preparation being ad- ministered to two rabbits. Blood was drawn before and at weekly intervals after the inoculation. Six weeks after the initial inoculation the rabbits were rechallenged with the same dose, and again bled weekly for three weeks.

Enzynze irnmunoussuys ( EIAs) In preliminary tests, optimal dilutions of reagents

for use in EIA were determined by box titrations. The dilutions selected were the highest that gave maximal absorbance values in EIA.

All EIAs were performed in 96-well microtitre plates (Maxi-Sorp, Nunc, Roskilde, Denmark). The antigens used for coating were either sonicated anti-

gens at a concentration of 5 or 10 pg protein per ml or whole-cell suspensions (ODh50:1.5) in 50 mM sodium carbonate buffer, pH 9.6. The sonicated antigen solution was dispensed at 100 p1 per well and incubated overnight at 4"C, and the whole-cell sus- pension was dispensed at 50 1.11 per well, incubated for 30 min, and centrifuged at 2500 rpm for 15 min. After washing the plates, serial dilutions of rabbit sera in dilution buffer were incubated for 2 h at room temperature. Binding of antibodies to the antigen was visualized by means of a peroxidase-conjugated im- munoglobulin fraction from swine antiserum to rabbit immunoglobulins (P 217, Dakopatts, Glostrup, Denmark) diluted 1 : l O O O in dilution buffer. The peroxidase activity was visualized with o-phenylenedia- mine (0.04'Yn wiv), 4.2 mM H202 in staining buffer (0.1 M citrate-phosphate buffer, pH 5) . The differ- ence in OD490 and ODhs0 was read in a Thermomax microplate reader (Molecular Devices Co., Menlo Park, CA 94025).

Crossed irnrnunoelectrophoresis ( X I E ) The electrophoresis was performed as described in

( 5 ) except that pre- and postinoculation sera from

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E N G B E K & KOCH

rabbits inoculated with live R. henselue, R. quintuna and A . jd i s were added to the intermediate gel of the reference XIEs at a concentration of 40 pl cmp2.

R ES U LTS

The antibody development (Fig. 1) in rabbits inoculated with live R. henselae, R. quintuna and A . felis was studied in EIA with both sonicated and whole-cell coating antigens. Except that less regular titration curves were obtained with wholecell antigens, no differences in the measured antibody responses were found with the two types of antigen preparation. In one of each pair of rabbits infected with R. quintana and A . felis respectively, there was hardly any increase in OD values. In the remaining rabbits the OD values rose above the preinoculation levels at 2 weeks and continued to rise during the follow- ing 2 weeks, after which they seemed to level

out. After the second inoculation, OD values increased considerably in the rabbits that had responded to the first inoculation, whereas they showed only a modest rise in the two rabbits that had not responded to the initial inoculation.

The development of cross-reacting antibodies against R. henselue, R. quintuna and A . felis was studied by EIA using both heterologous bacterial sonicates and whole cells as coating antigens. Figs. 2, 3 and 4 show the antibody responses of one rabbit from each pair tested against sonicate of the immunizing agent and three related bacteria as coating antigen. In rabbit 2902 inoculated with R. henselae, no significant cross-reacting antibodies developed after the first inoculation, but when the rabbit was rechallenged cross-reacting antibodies developed not only against the heterologous Roch- alinmeu species, but also against Brucellu

Fig. 5. Development of antibodies in rabbit 2902 inoculated with R. hensclue, as determined by crossed immunoelectrophoresis. 20 pl R. henselue sonicate was electrophoresed against 750 pl rabbit anti-R. hensrlur s c w t n with (A) 200 p1 preinoculation serum or (B) 200 pl postinoculation serum added to the intermediate gel. Precipitin lines not influenced by the presence of postinoculation serum in the intermediate gel are indicated by their reference numbers.

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ROC'HALIMAEA A N D AFIPIA INFECTIONS IN RABBITS

Fig. 6. Development of antibodies in rabbit 2904 inoculated with R. quintunu, as determined by crossed immunoelectrophoresis. 20 p1 R. quintmu sonicate was electrophoresed against 750 p1 rabbit anti-R. quin tun~~ serum with (A) 200 p1 preinoculation serum or (B) 200 p1 postinoculation serum added to the intermediate gel. Precipitin lines not influenced by the presence of postinoculation serum in the intermediate gel are indicated by their reference numbers.

abortus. In rabbit 2904 inoculated with R. quintana, low-titred antibodies also developed against A . felis after the initial inoculation, but not against the other bacterial species. After reinoculation, cross-reacting antibodies were produced against all three heterologous bacterial species tested. In rabbit 2907 inoculated with A . felis, no cross-reacting antibodies developed after the first inoculation, but antibodies cross- reacting with R. quinlanu antigens were produced after reinoculation.

Antigens of R. henselue, R. quintuna and A . felis against which antibodies were produced after inoculating rabbits with live bacteria were studied by means of XIE with pre- and postinoculation sera incorporated into the intermediate gel. After only one week the postinoculation sera produced deflection of some of the reference precipitin lines, and a maximum number of deflections was obtained after a fortnight.

The lines first affected were RH-9, RH-22, RH- 23, RH-31, RH-40, RH-48 and RH-51 after inoculation with R. henselue, RQ-9, RQ-26, RQ- 30, RQ-44 and RQ-49 after R. quintana, and AF-5, AF-12, AF-26, AF-28 and AF-33 after A . felis. After reinoculation, several more precipitin lines were deflected towards the intermediate gel and only 5-12 lines from each species remained unchanged. These non-reactive lines were RH-17, RH-21, RH-28, RH-44, RH- 47 and RH-53 for R. henselue, RQ-10, RQ-21, RQ-29, RQ-34, RQ-35 and RQ-48 for R. quintana, and AF-2, AF-6, AF-10, AF-11, AF-13,

and AF-39 for A . ,felis. AF-17, AF-19, AF-22, AF-32, AF-35, AF-36

DISCUSSION

The laboratory diagnosis of CSD and bacillary angiomatosis by culture is difficult. Conse-

947

ENGBEK & KOCH

Fig. 7. Development of antibodies in rabbit 2907 inoculated with A . felis, as determined by crossed immunoelectrophoresis. 20 pI A. felis sonicate was electrophoresed against 750 pl rabbit anti-A. fc~lis serum with ( A ) 200 p1 preinoculation serum or ( B ) 200 p1 postinoculation serum added to the intermediate gel. Precipitin lines not influenced by the presence of postinoculation serum in the intermediate gel are indicated by their reference numbers.

quently, serological testing is an attractive alter- native procedure for detecting these diseases. With the aim of investigating antibody responses to the postulated causative organisms of these diseases in rabbits we inoculated three pairs of rabbits with live R. henselue, R. quintunu and A. ,filis respectively, and bled them weekly. Six weeks after the initial inoculation the rabbits were rechallenged and bled again weekly for two weeks. In two of the rabbits the antibody titre did not rise after the initial inoculation, whereas four rabbits developed low antibody titres against the immunizing bacteria. The two rabbits that did not respond to the initial inoculation developed only a modest rise in antibody titre after reinoculation, whereas the rabbits that responded to the first inoculation showed a considerable rise in antibody titre after rechallenge. This observation is in agree-

ment with the common observation (e.g. the response of guineapigs to diphtheria toxoid or of mice to sheep red cells) that antibody production is under genetic control and that some animals are poor antibody producers while others form abundant amounts (2, 9).

A potential problem in serodiagnostic assays using bacterial sonicates or whole cells as antigens is the specificity of the test. If the objective is to identify a positive serum by looking for an elevated antibody titre, this may not be attained if there are cross-reactions with antigens from other bacteria. To assess the specificity of EIA in the serodiagnosis of R. henselue, R. quintunu and A. felis infections, we tested bacterial sonicates and whole bacterial cells of three related bacteria against serum of rabbits inoculated with live R. henselue or R. quintuncr or A. ,fdis. No significant or only low-titre cross-reactions

948

ROCHALIMAEA AND AFIPIA INFECTIONS IN RABBITS

were detected after the initial inoculation, but after rechallenge serum from rabbits inoculated with R. henselae or R. quintana cross-reacted with A . felis and Brucella abortus, and serum from the rabbit infected with A . felis cross-reacted with R. quintana, but not with R. henselae or B. abortus.

No studies have been published on R. henselae, R. quintana and A . felis infections in rabbits. None of the rabbits infected with one of the three bacteria appeared ill or developed signs of infection, and the bacteria probably died soon after the inoculation. The antibody titre which developed after such a short infection was probably too low to cause any cross- reaction with related bacteria. After rechallenge the antibody titre increased significantly and now a cross-reactivity between Rochalimaea/ Afipia and to a lesser extent between Rochali- maea/Brucella became evident. Thus it was im- possible to identify the infective organism in rabbits by looking for a rising antibody titre by EIA without also comparing the titre against related bacterial species.

The antibody response was also studied in XIE with pre- and postinoculation sera from the weekly bleeds incorporated into the intermediate gel. These studies showed that antibodies developed 7-14 days after inoculation and were directed against 5-7 antigens which in- cluded both specific antigens and cross-reacting antigens from related bacteria. After rechallenge, several more precipitin lines were deflected, including specific antigens (RH-45, RQ- 9, RQ-36, AF- 16 and AF-21), cross-reacting antigens from related bacteria (RH- 16, RH-30,

AF-18, AF-20 and AF-25) and common bacterial antigens (RH-52, RQ-15, RQ-46 and AF- 38), while only 5-12 precipitin lines were unchanged ( 5 ) .

Although the present findings have been based on experiments in laboratory animals they provide information on the time course of antibody appearance to the Rochalimaea/AJpia group of bacteria which is likely to be appli- cable to studies on human sera. Further investigation of the use of purified antigens in EIA

RH-49, RQ-30, RQ-36, RQ-44, RQ-47, AF-14,

for the diagnosis of these infections is now in progress.

We are grateful to Dr L. 0. Uttenthal for comments on the manuscript.

REFERENCES

1. Barka, N . E. , Hadfield, T. , Patniak, M.. Schwartzman, W. A . & Peter, J . B. : EIA for detection of Rochalimaeu henselae-reactive IgG, IgM, and IgA antibodies in patients with suspected cat-scratch disease. J. Infect. Dis. 167: 1503-1504, 1993.

2. Biozzi, G., Stiffel, C., Mouton, D., Bouthillier, Y. & Decreusefond, C.: Genetic selection for antibody production in mice. In: Peters, H. (Ed): Protides of the biological fluids. Pergamon Press, Oxford 1969, pp. 161-167.

3. Bradford, M . M.; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976.

4. Cooper, M. D., Hollingdale, M. R., Vinson, J . W. & Costa, J.; A passive hemagglutination test for diagnosis of trench fever due to Rochalimaeu quintana. J. Infect. Dis. 134: 605-609, 1976.

5. Engbak, K. & Koch, C.: Immunoelectrophoretic characterization and cross-reactivity of Rochali- maea henselae, Rochalimaea quintunu and AJipia felis. APMIS 102: 931-942, 1994.

6. Herrmann, J . E., Hollingdale, M. R.. Collins, M . F. & Vinson, J . W.: Enzyme immunoassay and radioimmunoprecipitation tests for the detection of antibodies to Rochalimaea (Rickettsia) quintuna. Proc. SOC. Exp. Biol. Med. 154: 285-288, 1977.

7. Hollingdale, M. R., Herrmann, J . E. & Vinson, J . W.; Enzyme immunoassay of antibody to Roch- alimaea quintana: diagnosis of trench fever and serologic cross-reactions among other rickettsiae. J. Infect. Dis. 137: 578-582, 1978.

8. Regnery, R. L., Olson, J . G., Perkins, B. A. & Bibb, W.: Serological response to “Rochulimaeu henselae” antigen in suspected cat-scratch disease. Lancet 339: 1443-1445, 1992.

9. Scheibel, 1. F.: Hereditary differences in the ca- pacity of guinea-pigs for the production of diphtheria antitoxin. Acta path. microbiol. scand. 20: 464-484, 1943.

10. Vinson, J . W. & Campbell, E. S.: Complement fixing antigens from Rickettsia quintana. Acta Vi- rol. 12: 54-57, 1968.

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antibody response in rabbits infected with rochalimaea henselae, rochalimaea quintana and afipia...

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