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Campylobacter fetus Diagnosis:Direct Immunofluorescence ComparingChicken IgY and Rabbit IgG ConjugatesAna Cipolla} , Jose Cordeviola', Horacio Terzolo', Gustavo Combessies' , Juan Bardon', Ramon Noseda',Alfredo Martinez', Dora Medina}, Claudia Morsella' and Rosana Malena''Institute Nacional de Tecnologia Agropecuaria, Estaci6n Experimental Agropecuaria de Balcarce, Departamento de ProduccionAnimal, Provincia de Buenos Aires, Argentina, 2Laboratorio Azul, Azul, Provincia de Buenos Aires, Argentina

SummaryIn Argentina Bovine Genital Campylobacteriosis is routinelydiagnosed by direct immunofluorescence test. Generally, thehyperimmune sera used for this test are obtained from rabbitsand less often from goats. In this work, a chicken egg yolkimmunoglobulin (IgY) extract was conjugated and its ability todetect campylobaeters with the regular conjugate preparedwith rabbit sera was comparatively evaluated. Both conjugateswere independently evaluated by two laboratories, named"Azul" (Lab A) and" Balcarce" (Lab B). Animals wereimmunised with formalin inactivated Campylobacter (C.)fetuscells. Chicken IgY and rabbit IgG were conjugated withfluorescein isothiocyanate and used to comparatively examinestrains ofC.fetus subspp., other Campylobacter spp. anddifferent bacterial species. Both conjugates had a highpercentage rate of detection for C.fetus. IgY had less back-ground due to unspecificfluoreseence than IgG. IgY is acheap, bloodless and very productive method.lgY can replacemammal immunoglobulins for C. fetus diagnosis.

Keywords: immunofluorescence, egg yolk, IgY, IgG, BovineGenital Campylobacteriosis Campylobacter fetus

1 Introduction

Bovine Genital Campylobacteriosis(BGC) is a disease generally caused byCampylobaeter (C.) fetus subspecies(ssp.) venerealis and occasionally by thessp.fetus (Dekeyser, ] 984; MacLaren andAgumbah, 1988). This disease is charac-terised by infertility and sporadic abor-tion (Garcia et al., 1983) and becausenatural breeding is commonly used, it isa widespread disease among herds from

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Zusammenfassung: Campylobacter fetus Diagnostik: Vergleichder Effektivitat von fluoreszenzmarkierten Huhn- (IgY) undKaninchen- (IgG) Antikorpern im direkten lmmunfluoreszenz-testDie bovine genitale Campylobacteriosis wird in Argentinienroutinemajiig durcn einen direkten Immunfluoreszenztestdiagnostiziert. Im Allgemeinen werden hierfiir Hyperimmunse-ra vom Kaninchen, seltener yon del' Ziege verwendet. In dervorliegenden Arbeit wurde die Leistungsfiihigkeit markierterIgY-Antikorper (prapariert aus Eidotter) vom Huhn mil denublicherweise eingesetzten Antikorpern vom Kaninchenverglichen. Hierzu wurden sowohl Huhner als auch KaninchenrnitFormalin-inaktivierten Campylobacter (C.) fetus Zellenhyperimmunisiert. Die erhaltenen Antikorper wurden mitFluoreszein-Isothiocynat markiert und zur Detektion yon C.fetus subspp., anderen c. spp. sowie davon unterschiedlichenBakterien eingesetzt. Eine Evaluierung beider Konjugateerfolgte unabhdngig in zwei unterschiedlichen Laboratorien,bezeichnet als "Azul" (Lab. A) und .Balcarce" (Lab. B).Beide Konjugate detektierten zu einem hohen Prozentsatz C.fetus. Allerdings zeichneten sich die IgY-Konjugate im Ver-gleich zu den IgG-Konjugaten (Kaninchen) durch cine deutlichgering ere unspezifische Hintergrundfarbung aus. Somit ist dieIgY-Technologie auch fur diesen Einsatzzweek eine tierscho-nende, kostengiinstige und sehr produktive Methode. Auf del'Basis der Ergebnisse diesel' Studie kann konstatiert werden,dajJfur die Diagnose von C. fetus herkommliche Kaninchen-Antikorper durch IgY-Antikorper ersetzt werden konnen.

Argentina (Villar and Spina, 1982; Russoet aI., 1998; Boullon et al., 2000). Due topractical and economical reasons, tradi-tionally the control of this disease mainlyrelies on the examination and vaccinationof bulls, also the carrier cow can main-tain the infection from one breeding sea-son to another (Cipolla et al., 1994). Atpresent its diagnosis is usually performedin males employing, as a screening teston preputial smegma, the direct immun-ofluorescence (DIP) test. Bacteriological

culture of BGC is an alternative method,though its cost and complexity determinesthat only few laboratories with appropri-ate infrastructure can routinely performit. DIP and culture can both be used todiagnose samples of preputial smegma,cervico-vaginal mucus and material fromaborted foetuses.InArgentina, mainly during 1983-92 the

number of veterinary laboratories work-ing on this disease has increased due tothe implementation of a National Sanitary

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Plan to control BGC and trichomoniasis(Terzolo et al., 1992; Dillon et al., 1997).At present, about 30 laboratories are en-gaged in the control of DGC and they rou-tinely employ the DIF lest in bull's screen-ing (Villar and Spina, 1982; Russo et al.,1998; Boullon et a!., 2000). As a directconsequence of the implementation of thisplan, the percentage of BGC infection inmeat herds decreased from around SO%in 1983 (CipoJla et aI., 1997) to aroundIS-18% in the period 1997-1999. ThisBGC infection decrease can be essentiallyattributed to the successful diffusion andimplementation of DIF screening test todetect infected bulls during 16 years. From1989 onwards, the Scientific Commissionon Bovine Venereal Diseases, dependenton the Argentine Association ofVeterinarians of Diagnosis Laboratories,recommended to the professionals incharge of the laboratories to participateeach year in a voluntary official control oftheir DIF technique and conjugates. Theprofessional staff of the National Instituteof Agricultural Technology (INTA), Ex-perimental Station of Balcarce, annuallyconducted the quality control. The partici-pating laboratories have annually carriedout the control tests from 1983 until to-day. During all these years, the number ofparticipant laboratories varied from 6 to19 and they have used from 7 to 3 differ-ent conjugates. All conjugates were elabo-rated using mammal hyperimmune sera,mainly prepared from rabbits and onlyonce a conjugate was elaborated from agoat serum. The diagnosis efficiency de-tected in these controls, using formalinfixed bacterial antigens and preputialwashings, improved through the yearsfrom 78% in 1991 up to 98% in 1996 andthe following years.

Considering that in our country conju-gates for DIF tests are essential for the con-trol of BGC and a significant number ofanalyses have to be annually carried outby different laboratories, quite a lot of rab-bits have to be killed every year to pre-pare the conjugates. As the specific henantibodies concentrate in the egg yolk at atitle similar to blood serum (Schade et al.,1996 and 2000) and separation egg yolkimmunoglobulins (IgY) from the lipids(Schwarzkopf and Thiele, 1996; Schadeet al., 2000) is now available, replacementof rabbits by hens is an alternative methodto be implemented. In fact, the hen is able

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to produce eggs containing specific 19Yagainst a great variety of bacterial antigens:C.jejuni and C. coli (Chandan et al., 1994);Escherichia coli (Yokoyama et al., 1992;Ikemori et al., 1993; Tanimoto et aI., 1995);Salmonella enterica serovar Enteritidis(Peralta et al., 1994; Terzolo et al., 1998);Brucella abortus (Losch et aI., 1986) andEdwarsiella tarda (Gutierrez et al., 1993).Besides, chicken IgY has been used toprepare fluorescent conjugates for thediagnosis of rabies, some avian viraldiseases (Staak, 1996) and Bordetellabronchiseptica (Hlinak et aI., 1996). Thus,a work to evaluate the ability ofIgY con-jugates to detect C.fetus sspp. strains wasundertaken.This study was carried out to induce an-

tibodies against C.fetus sspp. strains in lay-ing hens and rabbits, to extract specific IgYfrom egg yolks and to prepare and evalu-ate these two immunofluorescent conju-gates by two independent laboratories.

Unfortunately, for this study it was notpossible to use hen keeping equipment ac-cording to the standard usual in some Eu-ropean countries (at least in public insti-tutions). Therefore, the improvement ofkeeping conditions is one aspect of a jointIgY-project between Argentina and Ger-many which started in this year.

2 Animals, materials and methods

The rabbits and chickens were both sepa-rately kept using the available standard fieldrearing systems. All animals were rearedin cages to prevent coccidiosis and otherdiseases. The rabbits were individually al-located in outdoor cages. Rabbit cages are70 cm long, 80 em wide and SO cm high;they have cement roof and walls, wiredfloor and a double plastic mesh 2 metersover the cages to protect the animals fromthe sunlight. The hens were caged indoorsusing the available commercial wired bat-teries (41 em x 41 ern), originally designedto keep three hens per cage. All hens wereindividually caged so as to have plenty ofroom. The construction of the cages al-lowed the hens to have optical and acous-tical contact. Each cage has devices to feedand provide drinking water to the animals.All these cages are elevated from the floorso as the faeces drop on a cement floor.These faeces were daily collected, and thefloor cleaned and disinfected to minimisethe risk of infection with avian pathogens.

Animals were fed balanced food spe-cifically formulated for rabbits and lay-ing hens. Balanced foods were free fromantibiotics, coccidiostats and had only veg-etal ingredients, avoiding either meat orfishmeal. Batches of this balanced foodwere tested for detection of aflatoxins B 1,B2, GI and G2, ochratoxin A, T-2 toxinand zearalenone and found to be negative.Bacteriological analyses were made tocheck absence of coliform bacteria in wa-ter and Salmonella in water and food. Allanimals received water ad libitum. Rab-bits were fed ad libitum the balanced foodsupplemented with alfalfa (Medicagosativa) and hay and also have straw pro-vision for bedding. Each hen receiveddaily 100 grams of balanced food supple-mented with ground seashell. A lightingprogramme to increase egg productionwas adapted and the number of eggs wasrecorded daily.

To obtain specific serum antibodies, two8-week-old New Zealand rabbits receivedthree 0.5 ml subcutaneous applications ofa vaccine at days 0,33 and 72. Fifteen daysafter the last injection both rabbits werebled.

To obtain specific egg yolk antibodies,eight Golden Comet layers were immu-nised with two vaccines. Five layers wereimmunised with vaccine I and the otherthree hens with vaccine 2 (vide infra). Allhens were given two O.S ml doses into thepectoral muscles, separated by a IS-dayinterval. Nineteen days after the seconddose, all eggs were collected daily during3 days and stored at 4°C.

2.1 Bacterial strainsA total of 67 bacterial strains belongingto different genera and species were evalu-ated: Campylobacter (S7 strains) includ-ing C. fetus subspp. (53) and otherCampylobacter (4); Bacillus sp. (1); Bru-cella abortus (1); Proteus sp. (1); Actino-myces pyogenes (I); Escherichia coli (1);Salmonella enterica serovar Dublin (1);Staphylococcus aureus (1); Pseudomonasaeruginosa (1); Streptococcus sp. (1) andKlebsiella pneumoniae (1). Out of the S3strains of C. fetus subspp., 3S were C. fe-tus fetus (34 regional isolates and one in-ternational reference strain), 11 C. fetusvenerealis (10 regional isolates and oneof international reference strain) and 7 C.fetus venerealis biotype intermedius (allregional isolates).

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Each one of these strains was preservedin liquid nitrogen at -196°C. After thaw-ing, sub-cultures on Columbia agar, sup-plemented with 7% of bovine blood weremade. Incubation according to the tem-perature and atmosphere requirements ofthe different genera was carried out. Oncean abundant growth was obtained, thestrains were suspended in 1% formalinsterile PBS, pH 7.2. The concentration ofeach suspension was standardised toMcFarland's scale number 0.5 (l.5 x 108

cells per ml).

2.2 Vaccines for rabbitsThe vaccine was an equivalent mixture offive C. fetus strains: C. fetus venerealisNCTC 10354, C. fetus venerealis INTA371, C.fetus venerealis INTA 395, C.fe-tusfetus NCTC 10348, C.fetusfetus INTA96/567 and C.fetusfetus NCTC 5850. Thefinal concentration of the antigen mixturewas adjusted to 6 x 109 cells/ml by meansof the McFarland scale. One part of anti-gen, in a vehicle of 0.5% (v/v) formalinPBS pH 7.2, was mixed and emulsifiedwith one part of Freund's completeadjuvant.

2.3 Vaccines for chickensVaccine 1 was prepared with an equiva-lent proportion of three regional strains ofC.fetus venerealis, C.fetus venerealis bio-type intermedius and C. fetus fetus. Vac-cine 2 contained equal parts of 5 strains,including the three previously mentionedregional strains and two other regionalstrains of the subspecies venerealis andfetus. Both vaccines were inactivated with0.5% formalin and then 20% (v/v) alu-minium hydroxide gel containing 2% (w/v) of aluminium oxide was added asadjuvant. The final live bacterial concen-tration, determined by the method of Mileset al. (1938), varied from 1 to 6 X 109 CFU(average concentration per strain wasaround 1.3 x 109).

2.4 Rabbit serum IgG extraction andconjugationThe proteins were separated from the se-rum by precipitation with 70% (w/v) of(NH4)2S04' This solution was added at avolume identical to the original serum.After precipitation, the mixture was cen-trifuged and the supernatant was dis-carded. The sediment was suspended indistilled water at the same volume of the

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original serum. After the first (NH4)2S04precipitation, this procedure was repeatedtwice. Finally, the sediment was dissolvedin distilled water, using a volume of waterequal to half of the original volume of theserum. The resulting globulins were dia-lysed against a 0.85% NaCI solution untilno BaS04 precipitation was observed inthe solution when a 40% (w/v) of BaCl2was added. The amount of proteins in mgwas determined at 540 nm in a semiauto-matic SEAC spectrophotometer modelCH16 (Gornall et al., 1949). Globulinswere conjugated with a solution offluorescein isothiocyanate in N a2HPO 4 pH9, at about 25-30 ug per mg of protein.Next the conjugate was centrifuged at2,800 g during 20 minutes. Thereafter thesupernatant was dialysed at 4°C, againstPBS pH 7.6, until the dialysis solution re-mained clear. Finally 0.02% (w/v) N3Nawas added.

2.5 Chicken egg IgY extraction andconjugationA dozen egg yolks from hens of bothgroups, free from their membranes and ofalbumin, were pooled together, mixed andhomogenised (Schade, 2000). To obtainhigh purity IgY from fresh yolks, thepolyethyleneglycol extraction method ofPolson et al. (1980) was used(PROMEGA® kit "EGGstract IgY purifi-cation System"). Three precipitations weremade according to the manufacturer'sinstructions. Afterwards the same proce-dure previously described for the rabbitserum was carried out, including twoprecipitations with 70% (NH4)2S04(Schwarzkopf and Thiele, 1996), dialysisagainst PBS pH 7.6 and conjugation withfluorescein isothiocyanate. This extractwas also preserved with 0.02% (w/v)N3Na.

2.6 Participant laboratoriesTwo laboratories of veterinary diagnosis,located in the Southeast Region of Bue-

nos A ires Prov ince, carried out this work,These laboratories were the private Labo-ratory Azul (Lab A) and the official Labo-ratory INTA Balcarce (Lab B). Both labo-ratories routinely use the DIF techniqueto detect C.fetus in bulls.

2.7 Immunofluorescence techniqueThe technique used by both laboratorieswas previously described (Dufty, 1967)except for the fixation step at Lab A. Inthis laboratory the procedure was modi-fied fixing the slides by passing them 3times slowly through the flame (Kita etal., 1966). Then, slides were incubatedovernight at 37°C. Conjugates were di-luted at the 1:20 with PBS, pH 7.2.

2.8 Evaluation of the conjugatesBoth laboratories used the same methodto evaluate the conjugates. Each labora-tory worked with aliquots of the sameformalinised antigens and twice two dif-ferent operators observed each strain. Aresult was considered to be positive onlyif fluorescence was detected together withmorphology compatible to the C. fetus.Different degrees of fluorescence wereobserved and recorded as follows: weak:+; good: ++; very good: +++. Strains wereconsidered to be negative either if theydid not show any fluorescence or the mor-phology did not correspond to c. fetus.Lab A and Lab B respectively used Carl-Zeiss and Nikon epifluorescence micro-scopes. Both microscopes worked undermercury vapour lamp.

3 Results

C. fetus strains were detected at a highrate by both conjugates, although morestrains were detected by means of the rab-bit conjugate (see Table 1). Using the IgYconjugate in Lab A only one C.fetus straincould not be detected whereas in Lab Btwo strains were missed. On the otherhand, all C. fetus strains examined were

Lab

Tab. 1: C. fetus Lab A and B detection rates comparing chicken and rabbit conjugates

Chicken IgYN° and % of strainsdetected/evaluated

A

B

52/53 (98,1)

51/53 (96,2)

Rabbit IgGN° and % of strainsdetected/eval uated

53/53 (100)

53/53 (100)

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detected when the rabbit conjugate wasused.When these data were analysed compar-

ing the different C. fetus subspecies (seeTable 2), both laboratories failed todetect a single identical C. [etus fetusregional strain and only Lab B skipped thedetection of a reference strain of C.fetusvenerealis NCTC 10354.

could greatly reduce the number ofCampylobacter cells. Contamination ofpreputial samples is more frequent thanthose coming from vaginal mucus oraborted foetuses (Campero et al., 1993).DIF have the advantage to allow a rapiddiagnosis, even in the presence of heavycontaminant flora of the preputial envi-ronment. Therefore the DIF technique is

Tab. 2: C. fetus subspp. Lab A and B detection rates comparing chicken and rabbitconjugates. Cfv: C. fetus veneralis, Cfvi: C. fetus veneralis biotype intermedius, eft: C.fetus fetus

Lab Chicken IgY Rabbit IgGNQand % of strains NQand % of strainsdetected/evaluated detected/evaluated

Cfv* Cfvi** Cff*** Ctv Ctvi Cff

11/11 7/7 34/35 11/11 7/7 35/35(100) (100) (97,1) (100) (100) (100)

10/11 7/7 34/35 11/11 7/7 35/35(90,9) (100) (97,1) (100) (100) (100)

A

8

Using both conjugates, none ofthe other14 bacterial species examined, all differ-ent from C. fetus, showed any fluores-cence. Therefore the specificity of thechicken and rabbit conjugates was 100%at both laboratories.Interestingly, in comparison with the

IgG conjugate, all antigens analysed withthe IgY conjugate showed lower unspe-cific background fluorescence.

4 Discussion

Bacteriological culture and DIF have beenconsidered traditional methods for C.fe-{US diagnosis. Using these techniques, thedetection rates differ according to the re-ports of different authors. Thus, for cul-ture detection, percentages near to orhigher than 90% have been registered(Ardrey et al., 1972; Palladino andCampero, 1983; Hum et al., 1994)whereas for DIF the values have oscil-lated between 60% (Philpott, 1968) and90% or even more (Mellick et al., 1965;Dufty, 1967).When the isolation technique is used,

it is essential to control the overgrowthof C.fetus by contaminant bacteria, suchas Pseudomonas and Proteus genera. Toavoid contamination, filtration of samplesmay be done, although this procedure

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a good procedure for the diagnosis ofBGC in bulls, although it critically de-pends on the employment of reliable con-jugates. These conjugates have to be spe-cific and sensitive enough to respectivelyavoid detection of false positive and nega-tive results. To that end, it is required tohave both excellent immunofluorescentconjugates and high-quality optic micro-scope equipment, so as to achieve maxi-mum sensitivity and specificity. In Argen-tina, conjugates for the BGC diagnosishave been commercialised for manyyears, being basically elaborated in mam-mals, and mainly in rabbits, followingpre-established standard protocols(Mellick et a1., 1965; Philpott, 1968).This work compares the performance

of a commercial rabbit conjugate, rou-tinely used for diagnosis in Argentina,with an IgY conjugate particularly pre-pared for this experimental study. It wasdemonstrated that usage of incompleteFreund's mineral oil adjuvant in hens gen-erally provides a better level of specificantibodies due to production of sterileabscesses, which increases the depot ef-fect (Schade et a1., 2000). In this workwe used aluminium hydroxide geJadjuvant because it was ready availablein our laboratory and, in our experience,it produces a good immune response with

bacterial antigens. Future work has toconsider also the usage of Freund'sadjuvant by subcutaneous route (Schadeet al., 2000).The sensitivity of both conjugates was

high, being 100% for the rabbit conjugateand 96.2% - 98.1% for the IgY conjugate.These differences may be due to: usageof different optic equipment in both labo-ratories, loss of antigens during subcul-tures, antigenic differences among thestrains, and chiefly the inclusion of dif-ferent strains in the vaccines for rabbitsand in hens. Future work has to includeseveral representative regional isolates inthe elaboration of vaccines. In this work,when a c. fetus fetus regional strain wasnot included in the vaccine for hens, theIgy conjugate consistentl y skipped detec-tion of this strain. This DIF negative re-sult demonstrates antigenic differenceamong C.fetus strains. This phenomenon,which appears infrequently, was also re-corded with the rabbit commercial antis-era used in diagnosis cases in Argentina.It was demonstrated that the surface pro-tein antigens of C. fetus, conforming themicrocapsule, could be lost by severalsubcultures (Winter et a1., 1978; Cipollaet al., 1992), so next works have to con-sider not only the selection of adequatestrains but also their proper maintenance.Other factors, such as dexterity of the

operator in the reading of the slides anddilution of the conjugate are omitted,since our trained technicians have beenperforming this routine diagnosis taskduring over 20 years and all conjugateswere equally diluted.These preliminary results support fu-

ture work, taking into account the follow-ing items: use of identical bacterial anti-gens in the formulation of the vaccinesfor rabbits and hens, evaluation of a largenumber of strains and also to include sam-ples from infected cattle.In comparison with the IgG conjugate,

a lower unspecific fluorescence was ob-served with the IgY conjugate in all thesamples examined. This fact could be ex-plained because the IgY conjugate waselaborated in hens, an animal which isphylogenetically more distant from thebovine species than the rabbit (Shade etal., 2000). This property of IgY conju-gates is crucial when samples containingmammal organic material, such assmegma or preputial washings, are micro-

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scopically observed. Therefore replace-ment of rabbits by hens could improve thequality of conjugates for the diagnosis ofBGC and also may improve the DIP di-agnosis in other bacterial diseases.

Thus, the use of hens to produce spe-cific antibodies is a real alternative to theuse of rabbits from a view of animal wel-fare, science and also economy.

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serovariedad Enteritidis. Rev. Arg.Microbial. 30. 84-92.

Terzolo, H. R., Argento, E., Catena, M. C.et al. (1992). Procedimientos deIaboratorio para el diagn6stico de lacampy lobacteriosis y tricomoniasisgenital bovina. In Documento de LaComision Cientifica permanente deEnfermedades Yenereas de los bovinos(1-33). Balcarce,Argentina: CERBAS-INTAPress.

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AcknowledgementsThe authors thank Dr. Rudiger Schade,from the Institute of Pharmacology andToxicology, Department of Medicine

(Charite), Humboldt University, Berlin, forthe critical reading of this manuscript. Thiswork was supported by both The Special-ised Veterinarian Diagnostic group of TheNational Institute of Agricultural Technol-ogy (INTA), Department of Animal Pro-duction, Experimental Station of BalcarceandAzul Diagnostic Laboratory,Argentina.

Correspondence addressDr. Ana CipollaDepartamento de Producci6n AnimalINTA EEA BalcarceCC 276B7620EMA BalcarceProvincia de Buenos Aires/Argentina

Linz 2000, Poster Nachtrag

PosterIm Heft 1/2001 fehlte bei der Berichterstattung iiber Linz 2000 leider dieses Poster.ALTEX bittet urn Entschuldigung.Evaluation of Nutrition Media Derived from HumanBlood Transfusion Units DFP, EC and BC for the invitro Feeding of pediculus humanus corporis(Anoplura: Pediculidae)Birgit Habedank' and Gabriele SchraderI Free University Berlin, Institute for Parasitology and International Animal Health,2 Federal Environmental Agency, D-Berlin

The laboratory rearing of the human bodylouse Pediculus (P.) humanus corporis,main vector of Rickettsia prowazecki,Borrelia recurrentis and Bartonellaquintana, is unalterably required for effi-cacy tests of pediculicids. Beside the prac-tised for decades feeding procedure of thebody lice on arms of volunteers,Culpepper succeeded for the first time in1948 in breeding these obligate haemat-ophagous and stenoxene human parasitesexclusively by blood feeding on rabbits.Until now, rabbits are used as substitutehosts for the laboratory rearing of P.humanus corporis. However, the perform-ance of feeding lice on rabbits repeatedlygave occasion for criticism from point ofanimal welfare. Hence, there is a demand

170

for the development and evaluation of al-ternative methods for continuous labora-tory rearing and mass breeding of the hu-man body louse.Experimental investigations on the in vitrofeeding of P. humanus corporis of a rab-bit-adapted laboratory strain showed thatthis Anoplura species are able regularlyto feed on heparinised and stored at 4°Cfresh blood of volunteers, as well as onwhole blood resuspended from humanblood transfusion units (superimposedCPD-SAGM-stabilisised erythrocyte con-centrate unit - EC, superimposed deep fro-zen Aphaeresis-fresh plasma unit - DFPand a buffy coat unit - BC) that were of-fered through a Parafilm M®membrane(American National Can, Chicago). Thus,

the body lice were kept in laboratory over9 generations by continued in vitro feed-ing with blood stored until use at -27°C.However, a developmental deceleration ofI to 2 days from the first feeding of firststage larvae until their moult into imag-ines of in vitro fed lice compared to rab-bit fed lice was observed. Screenings of13 nutrition media (storage 4°C vs. -27°C) derived from Ee, DFP and BC andof 5 nutrition media extracted from BCin continued up to 30 days feeding ex-periments indicated that superimposedmedia from BC: GFP in ratios of 2:3, 1:1und 3:2 are proper for long time feedings.After the use of BC-derived nutritionmedia P. humanus corporis showed thesame developmental time from first blooduptake of first stage larvae to their moultinto imagines as lice fed on the rabbits.This study demonstrates that P. human uscorporis can be reared by membrane feed-ing technique. The in vitro feeding of hu-man lice offers the possibility of breed-ing these pediculids in considerateness toanimal welfare for studies on lice controland efficacy tests of insecticides as wellas for molecular biological and physi-ological studies. On the other hand thisin vitro method enables to offer the licedefined and standardised nutrition mediafor advanced studies.

ALTEX 18,3/01