Serological diagnosis of Neospora caninum infection

Serological diagnosis of Neospora caninum infection

C. BjoÈ rkmana, *, A. Ugglab

aDepartment of Ruminant Medicine and Veterinary Epidemiology, Swedish University of Agricultural Sciences, PO Box 7019,

SE-750 07 Uppsala, SwedenbDepartment of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences,

PO Box 7073, SE-750 07 Uppsala, Sweden

Received 26 April 1999; received in revised form 24 June 1999; accepted 30 June 1999

Abstract

Since the ®rst isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have beendeveloped for use in dogs, cattle and a variety of other potential host species. The tests include the indirect

¯uorescent antibody test, the direct agglutination test and di�erent enzyme-linked immunosorbent assays. Thisarticle reviews the principles and properties of the available tests which are discussed in relation to di�erentapplications. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: DAT; Diagnosis; ELISA; IFAT; Neospora caninum; Review; Serology

1. Introduction

The apicomplexan parasite Neospora caninumis closely related to Toxoplasma gondii, withwhich it shares many morphological and biologi-cal features [1±4]. However, N. caninum is, inprinciple, antigenically di�erent from T. gondii,an observation originally used in itsidenti®cation [1, 5]. The majority of N. caninuminfections appear to have a chronic course, prob-ably with life-long persistence of the parasite intissues of the infected individual. Similar to T.gondii, infection with N. caninum gives rise to anantibody response which can be demonstrated by

di�erent tests. The presence of antibodies in an

animal indicates that it is, or has recently been,

infected with the parasite. For general reviews on

the biology and clinical e�ects of N. caninum,

see [3, 6].

Development of a serological test requires

access to the particular infectious organism for

production of the assay antigen. In connection

with the ®rst successful isolation of N. caninum

from infected dogs by Dubey et al. in 1988, cell-

culture-derived organisms were used as antigen in

an IFAT [7]. An ELISA was described by

BjoÈ rkman et al. 1994 [8], and since then a rapid

expansion has taken place in the development

and application of di�erent ELISAs for the diag-

nosis of this organism. Recently, Romand et

al. [9] and Packham et al. [10] described a direct

agglutination test (DAT) which has the advan-

International Journal for Parasitology 29 (1999) 1497±1507

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(99 )00115-0

* Corresponding author. Tel.: +46-18-67-17-78; fax: +46-

18-67-35-45.

E-mailaddress:[email protected](C.BjoÈ rkman)

tage of not requiring species-speci®c secondaryantibodies.

The rapid increase in knowledge of the biologyand epidemiology of N. caninum would not havebeen possible without the employment of serolo-gical assays. This review will discuss availabletests for N. caninum antibodies, and their appli-cations in di�erent hosts.

2. Properties of serological tests

The ability of a test to distinguish betweeninfected and non-infected individuals is oftendescribed by its diagnostic sensitivity and speci-®city. The sensitivity is de®ned as the proportionof infected individuals correctly identi®ed by thetest, and the speci®city is the proportion of non-infected individuals correctly identi®ed [11]. Toevaluate the sensitivity and speci®city of a serolo-gical assay, sera from known infected as well asnon-infected animals need to be analysed. WithN. caninum, animals can harbour a latent infec-tion without showing clinical signs, and thismakes the de®nitive identi®cation of non-infectedindividuals critical [12, 13].

The sensitivity and speci®city of a test can bemanipulated by choosing di�erent cut-o� levels,i.e. values above which a result is deemed posi-tive. An alteration of the cut-o� value alwaysa�ects both the sensitivity and speci®city, e.g. anincreased cut-o� results in a higher speci®city,but also in a lower sensitivity. Depending on theapplication, either a higher sensitivity or speci-®city may be preferred.

Although the sensitivity and speci®city of aserological test are important features, cliniciansand epidemiologists are often more concernedwith the test's predictive value, i.e. the prob-ability that a test result re¯ects the true diseasestatus. The predictive value for a positive testresult is the probability that an animal isinfected, whereas the predictive value for a nega-tive result is the probability that an animal doesnot carry the infection. The predictive values of atest vary with the prevalence of the particulardisease in the population studied. As the preva-lence of infection decreases, the positive predic-

tive value decreases and the negative predictivevalue increases [11].

Newly introduced tests are often calibratedagainst an established reference method, regardedas a gold standard. The ®rst serological assay tobe developed for the demonstration of antibodiesto T. gondii was the dye test of Sabin andFeldman [14]. For a long time it was regarded asthe gold standard for T. gondii serology. The dyetest could be used in di�erent host species anddid not require species-speci®c secondary anti-bodies. For various reasons, an N. caninum dyetest has not been found feasible; instead, theIFAT has been regarded as a reference test withwhich other assays for N. caninum antibodieshave been compared and calibrated.

3. Indirect ¯uorescent antibody test

The IFAT was the ®rst serological test usedfor the demonstration of antibodies to N.caninum [7]. The test is based on the principle ofa�xing intact N. caninum organisms (tachyzoites)to microscopic slides which are incubated withthe diluted test serum and in a second step with¯uorescein-labelled antibodies directed againstimmunoglobulins of the animal species under in-vestigation. The reaction is evaluated under a¯uorescence microscope. For a positive result,bright, unbroken peripheral ¯uorescence of theorganisms is required. As in the T. gondii IFAT,sole ¯uorescence of the apical part of the tachy-zoites (`cap' or `polar' staining) is regarded as anon-speci®c reaction caused by infection withcross-reactive apicomplexan species [15, 16].Performance of the test requires training and ex-perience, and because of visual, individual in-terpretation of reactions, IFAT results are to acertain degree subjective. It is imperative thatthe optimal dilution of the ¯uorescein-labelledsecondary antibody (the conjugate) is carefullyestablished with known positive and negativecontrol sera along with the particular microscopeused. Reagents for N. caninum IFAT are com-mercially available (VMRD).

Because intact tachyzoites are used as antigenin the IFAT, the test mainly detects antibodies

C. BjoÈrkman, A. Uggla / International Journal for Parasitology 29 (1999) 1497±15071498

directed to antigens present on the cell surface ofthe parasite. With apicomplexan species, suchantigens are considered more speci®c than intra-cellular components [8, 10, 17]. Infection studiesin di�erent hosts have shown that the N. caninumIFAT shows very little cross-reactivity with othercoccidian parasites [18, 19]. This is particularlyimportant in relation to T. gondii. For thisreason, the IFAT is often used as a reference testfor N. caninum antibodies with which otherassays are compared.

The cut-o� titre employed in IFATs is depen-dent on a range of factors, such as conjugatecharacteristics and microscope properties, andcan be arbitrarily selected by varying the dilutionof the conjugate. Cut-o� titres in IFATs di�erbetween laboratories, but are often set to 1:50 incanine systems and 1:160±1:640 for bovines. Thedivergence in test performance between labora-tories makes direct comparison of IFAT titresdi�cult.

Neospora caninum tachyzoites of both canineand bovine isolates have been used as antigen inIFATs at di�erent laboratories. However, thereare no indications that possible minor antigenicdi�erences between isolates a�ect the accuracy ofthe test. Nor does it seem that antigen derivedfrom a bovine isolate is preferable for analysis ofbovine samples, or similarly to canine-derivedantigen for demonstration of antibodies in thedog. The N. caninum IFAT has been used todetect antibodies from a large number of animalspecies, including dog, fox, cat, cattle, sheep,goat, water bu�alo, horse, rodents and primates.

4. Direct agglutination test

A T. gondii DAT was described in 1959 byFulton and Turk [20], and was further developedby Desmonts and Remington [21]. It has sincebeen one of the most widely used serologicaltests for T. gondii in both human and animal sys-tems, and is commercially available as a test kit(Toxoscreen; bioMeÂ rieux). The principle of theDAT is that intact formalin-treated tachyzoitesagglutinate in the presence of speci®c antibodies.The modi®ed test detects only IgG antibodies,

because both speci®c and non-speci®c IgM isdestroyed by mercaptoethanol which is includedin the assay. For performance of the test, largenumbers of tachyzoites are needed so that thereaction on the bottom of microtitre wellsbecomes visible by the naked eye. In 1998, twoindependent papers described N. caninum DATsaccording to the principles of the modi®ed T.gondii DAT [9, 10]. The test described byRomand et al. [9] employed tachyzoites of thecanine NC-1 isolate, whereas that of Packham etal. [10] used tachyzoites of the bovine BPA-1 iso-late. When a large number of sera from up to 16di�erent animal species were analysed and resultscompared with those of the IFAT, the DAT wasfound to have a sensitivity and speci®city com-parable with those of the IFAT [10]. Also, thetest proved highly speci®c when sera from ani-mals infected with related parasites, including T.gondii, were tested [9, 10]. Because of its simpli-city and versatility, the N. caninum DAT has thepotential of being used for analysis of sera froma variety of species, thus possibly replacing theIFAT as ®rst-hand test in many situations.However, as with all serological tests, its applica-bility in di�erent host systems has to be carefullyevaluated. The N. caninum DAT has recentlybecome commercially available (VeÂ toquinol).

5. Enzyme-linked immunosorbent assay

5.1. Assay principles

5.1.1. Indirect ELISAThe ELISA principles are widely applied for

the demonstration of antibodies directed to avariety of infectious agents, including T. gondiiand N. caninum [22]. The particular antigenpreparation is coated onto the plastic surface ofmultiwell microtitre plates. After incubation withthe diluted sera to be analysed, an enzyme-labelled, species-speci®c anti-immunoglobulinantibody (conjugate) is applied. In a ®nal step asubstrate is added which, in the presence of theconjugate, is transformed to a coloured product.After a ®xed time the enzyme±substrate reactioncan be stopped before the absorbance or O.D. is

C. BjoÈrkman, A. Uggla / International Journal for Parasitology 29 (1999) 1497±1507 1499

measured by a spectrophotometer. Alternatively,the change in absorbance can be measured and amaximum slope of O.D. over time (Vmax) calcu-lated. The ®rst of these procedures is the mostwidely adopted, but Vmax values were used inone of the published N. caninum ELISAs [23].Also, the sera under investigation can be seriallydiluted and the ELISA results communicated astitre values.

Traditional indirect T. gondii and N. caninumELISAs utilise crude tachyzoite preparations con-taining a large number of antigens, the majorityprobably being of intracellular or cytoplasmicorigin. However, the speci®city and sensitivity ofT. gondii assays based on intracellular antigenshave been questioned [17], and it has beensuggested that serological tests based on surfacemembrane antigens should be more parasite-species-speci®c [24, 25]. To limit the exposure ofintracellular antigens, ®xed whole tachyzoites canbe coated onto the microtitre wells [26]. Parasitemembrane antigens can also be enriched and in-corporated into so-called immune stimulatingcomplexes (iscoms) to be used in the assay [8, 27].Other ways to increase the speci®city of the testhave also been tried. These include the reductionin number of antigens by using de®ned recombi-nant proteins [28, 29], or to capture a speci®cantigen by a mAb [30]. All these types of antigenpreparations are currently used in N. caninumELISAs at di�erent laboratories, and will befurther discussed below.

The most commonly used secondary antibodiesare di�erent polyclonal antisera directed againstimmunoglobulins of the animal species investi-gated. However, in some of the published N.caninum ELISA systems, mAbs to speci®c immu-noglobulins have been used to enhance the sensi-tivity and speci®city of the assay [8, 26, 31]. TheELISA has the advantage over IFAT that theregistration of reactions is done objectively andthe assay can easily be automated. It is thereforea technique well-suited for screening of largenumbers of samples, e.g. in serological surveys.

5.1.2. Competitive ELISAsCompetitive ELISAs are indirect tests in which

a mAb is included and allowed to compete with

antibodies in the test serum for available epitopeson the assay antigen. The secondary antibody isdirected to mouse immunoglobulins, i.e. themAb. If the test serum contains antibodies di-rected to the same epitope as the mAb, theabsorbance will be lower in wells containing amixture of mAb and test serum than in wellswith only mAb. The result is most often pre-sented as percentage inhibition by the test serum.One advantage with a competitive ELISA is thatit estimates the level of antibodies to one singleepitope, and it is thus likely to be more speci®cthan conventional ELISA systems. Furthermore,except for the anti-mouse antibody, no species-speci®c conjugates are needed, which makes acompetitive ELISA highly versatile. The competi-tive ELISAs for N. caninum antibodies in cattlethat have been presented utilise a mAb directedto a 65-kDa N. caninum surface antigen [30, 32].

5.2. Approaches in N. caninum ELISAs

5.2.1. Crude antigen ELISADi�erent crude antigen preparations containing

a mixture of intracellular and membrane antigenshave been used in N. caninum ELISAs. Whole-tachyzoite lysates have been prepared either bysonication [23] or by detergent solubilisation [33].A water-soluble extract was prepared by freezingand thawing tachyzoites followed by sonicationand removal of non-soluble material bycentrifugation [34]. Indirect ELISAs utilisingcrude antigen preparations of the bovine isolateBPA-1 and the canine NC-1 isolate have beendescribed in detail [23, 33, 34]. PareÂ et al. [23]reported 89% sensitivity and 97% speci®city fora bovine whole-tachyzoite lysate ELISA. Otherlaboratories report sensitivities and speci®cities of92±98% and 87±100%, respectively, for crudeantigen ELISAs [33±35].

One serum from a Babesia bovis-infectedcalf reacted in one of the lysate ELISAs [33],but otherwise no signi®cant cross reactivitieswith related protozoa, including T. gondii, havebeen described. Crude extract ELISAs havebeen adopted for use in cattle, sheep andgoats. An ELISA-kit for bovine serum utilisinga whole tachyzoite lysate is commercially


available (HerdChek Anti-Neospora; IDEXXLaboratories).

A whole-tachyzoite lysate was used as antigenin the competitive ELISA described by Baszler etal. [32]. The sensitivity and speci®city for this testwere not given, but all sera that were positive byIFAT inhibited mAb binding by more than 60%.Negligible binding was seen with some sera fromT. gondii- and Sarcocystis cruzi-infected animals.

5.2.2. Fixed tachyzoite ELISAIn a method described by Williams et al. [26],

puri®ed tachyzoites of the N. caninum NC-Livstrain were coated onto microtitre plate wells and®xed with 4% bu�ered formalin. This treatmentmakes only the surface membrane antigens acces-sible for antibodies in the test serum. mAb tobovine IgG was used as the conjugate. No cross-reactivity was found with sera from cattleinfected with T. gondii, S. cruzi, Eimeria spp.,Cryptosporidium parvum or Babesia divergens.When compared with the IFAT, the sensitivityand speci®city of this ELISA were reported to be95% and 96%, respectively. After slight modi®-cation the assay has been commercialised(Mastazyme-Neospora; MAST Diagnostics).

5.2.3. Iscom ELISAA way to limit the number of cytoplasmic pro-

teins is to incorporate N. caninum membraneantigens into iscoms, which are cagelike struc-tures of about 40 nm composed of Quil A, cho-lesterol, phospholipids and antigen [8, 31, 36].The iscom concept is thus used as a tool to selectfor amphipathic antigens, such as tachyzoitemembrane proteins [37]. Characterisation studieshave shown that N. caninum NC-1 iscoms con-tain membrane antigens from both the cell sur-face and from intracellular compartments. Themajor antigens included have approximate Mr of18, 30±45 and 61 kDa [8, 38]. The ®rst publishedELISA for demonstration of N. caninum anti-bodies was an iscom ELISA developed for analy-sis of canine sera [8]. Later, an iscom ELISA foruse with bovine serum as well as milk wasdescribed [31], and it has also been modi®ed toenable analysis of sera from water bu�aloes [39]and sheep (C. BjoÈ rkman, unpublished data).

Both the canine and bovine iscom ELISAs utilisemAbs, to canine Ig and bovine IgG1, respect-ively.

No cross-reactivity with T. gondii, or otherclosely related protozoa, was observed with theiscom ELISA. The sensitivity and speci®city werecalculated using IFAT as a reference method.For the dog assay, the sensitivity and speci®citywere 98% and 96%, respectively, whereas thecorresponding ®gures for the bovine ELISA were100% and 96%.

In epidemiological investigations of bovineneosporosis, it is often desirable to distinguishbetween acute and chronic infections. The levelof N. caninum IgG antibodies, or demonstrationof rising antibody titres, cannot be used for thispurpose, as these antibodies can persist at highlevels for long time and can ¯uctuate duringpregnancy [40]. The iscom ELISA has beenmodi®ed to enable analysis of IgG avidity (func-tional a�nity) [41]. Avidity assays are based onthe fact that the ®rst antibodies synthesised afterantigenic challenge or primary infection have alower a�nity for the antigen than those producedlater on. The N. caninum IgG avidity ELISA hasnot yet been fully evaluated, but the results sofar indicate that it is able to discriminate betweenacute and chronic infections.

5.2.4. Recombinant protein ELISASeveral laboratories are screening cDNA

libraries for clones that express proteins, with po-tential for use in future vaccines or assays for N.caninum infection. A number of such proteinshave been tested, but only four ELISAs based onrecombinant proteins have been published todate. Lally et al. [28] used, separately, two recom-binant proteins with Mr of 30 and 35 kDa asantigen in ELISA. When testing sera fromacutely infected cattle, there was a fairly goodagreement between the two tests and IFAT.Later, it was shown that antibodies to theserecombinant proteins were demonstrable in cowsfor at least 1 year after aborting N. caninuminfected foetuses [42]. Now a mixture of the tworecombinant proteins is used as ELISA antigenin their laboratory. Louie et al. [29] evaluated theusefulness of two protein fragments of 20 and


29 kDa in two di�erent ELISAs. They found aconsiderably higher sensitivity when either of thetwo recombinant proteins was used rather than awater-soluble extract of N. caninum tachyzoites.

5.2.5. Antigen capture ELISAA capture ELISA in which a mAb was used to

bind a 65 kDa N. caninum protein onto themicrotitre plate was applied by Dubey et al. [30].By the principles of a competitive assay, theserum sample and, thereafter, an enzyme conju-gated mAb directed to another epitope of the65 kDa antigen, were allowed to react with thecaptured protein. No speci®city or sensitivitydata were given for this ELISA.

6. Applications

6.1. Dog

After the discovery of N. caninum as a cause ofneuromuscular disease of dogs, the need for sero-logical tools for veri®cation of a clinical diagno-sis became evident. An IFAT employing NC-1isolate tachyzoites as antigen was used for thedemonstration of antibodies to N. caninum indogs by Dubey et al. [7] and in a number of con-secutive papers; however, the performance of thetest was not described in detail. In a study byBarber and Trees [13], blood dried onto ®lterpaper as well as canine colostrum and milk weresuccessfully used as sources of antibodies andtested by IFAT. Serological screenings of N.caninum infection in foxes and Australian dingoeswere performed by IFAT, utilising antibodies todog IgG as conjugate [43, 44].

The only ELISA described for use in dogs isthe iscom ELISA [8]. This test had a sensitivityand speci®city of 98% and 96%, respectively,against the IFAT as indicator of true status. Forscreening large numbers of sera, ELISAs areusually time and cost e�ective. However, atveterinary diagnostic laboratories usually fewsamples from dogs are submitted for Neosporaserology at any one time. Therefore, because ofits ¯exibility, most laboratories use the IFAT forroutine N. caninum serology. If the N. caninum

DAT [9, 10] proves reliable after further evalu-ation, it may replace the IFAT for routine diag-nosis of neosporosis in dogs.

6.2. Cattle

Since the presence of N. caninum in an abortedbovine foetus was ®rst reported in 1989, many in-vestigations of the occurrence and relative im-portance of neosporosis in cattle have beenperformed. An IFAT based on the bovine BPA-1isolate was the ®rst serological test to be usedwith cattle sera [45], and IFAT has since beenused in many studies of bovine N. caninum infec-tion (see reviews [3, 6]). As both diagnostic inves-tigation and research on N. caninum in cattleoften involve large numbers of sera, ELISA hasreplaced IFAT as the predominant serologicalmethod. Crude extract ELISAs are most widelyadopted [35, 46, 47], but the iscom and the whole-cell ELISA have also been used in epidemiologi-cal studies [12, 48]. The bovine iscom ELISA [39]and the DAT [49] were applied in the analysis ofsera from water bu�aloes.

In cattle, N. caninum antibodies ¯uctuateduring pregnancy, and they may, at least withsome assays, even drop below detectionlimits [45, 48]. Few attempts have been made tocompare and critically evaluate the di�erent sero-logical methods currently in use for bovines. Inone study, sera from cows in a dairy herd experi-encing an N. caninum abortion outbreak wereanalysed by several methods. These were IFATand ®ve ELISAs, including two di�erent tachy-zoite lysate ELISAs, the iscom ELISA, a recom-binant protein ELISA and an antigen captureELISA [30]. With exception of the recombinantprotein ELISA [42], there was a good agreementbetween the tests in di�erentiating between nega-tive and positive sera, but there was a poor quan-titative correlation in titre or absorbance values.However, very few non-infected animals wereincluded in the study and the majority of seratested were positive in all the assays. Schares etal. [50] reported a higher sensitivity for a crudeantigen ELISA (HerdChek Anti-Neospora) thanfor IFAT. Sera that tested positive in the ELISAalso reacted speci®cally in Western blot immu-


noassay, indicating that the discrepancy betweenthe ELISA and IFAT was not a result of poorspeci®city of the ELISA. The high sensitivity of acrude antigen ELISA was also demonstrated byWouda et al. [33]. They also reported that thewhole-tachyzoite ELISA (Mastazyme-Neospora)was less e�ective in detecting low antibody levelsin some chronically infected cattle.

Barr et al. [51] investigated the potential offoetal serology as a tool to diagnose N. caninum-associated abortion. In cattle there is no transpla-cental transfer of antibodies to the foetus. Thus,if N. caninum antibodies are present in foetalserum, or body ¯uids, it means that the foetus isinfected. It was found that IFAT was highlyspeci®c for foetal N. caninum infection and infoetuses older than 6 months the sensitivity was79% [51]. Similar results were reported byWouda et al. [52], who also found that a recom-binant ELISA [42] was too insensitive for use infoetal serology. Using pleural ¯uid rather thanserum, Otter et al. [53] found that 21 out of 25aborted foetuses with con®rmed N. caninuminfection had IFAT antibodies. Thus, althoughthe presence of speci®c antibodies in a foetus in-dicates that it is carrying the parasite, the failureto demonstrate antibodies in a bovine foetusdoes not rule out the presence of an N. caninuminfection. Besides, serology is not applicable inyoung foetuses, because the bovine foetus is notcapable of producing antibodies in response toan antigenic challenge before about 5 months ofage.

6.3. Small ruminants

Neospora caninum was ®rst reported in conge-nitally infected lamb in 1990, but serological in-vestigation was not performed on thisretrospectively studied lamb [54]. The IFAT hassince been used to monitor the course of exper-imental ovine N. caninum infection [55, 56] andwas also applied for analysis of CSF [56].

In a Costa Rican dairy goat herd with con-®rmed foetal neosporosis, IFAT was used as acon®rmatory test and antibodies to N. caninumwere demonstrated in six out of 81 doesinvestigated [57]. No N. caninum antibodies were

found when 200 goat sera from Norwegian farmswith abortion problems were analysed byIFAT [58]. The DAT was used to con®rm thepresence of speci®c antibodies in a pygmy goatthat aborted an N. caninum infected foetus [10].

6.4. Horse

A few cases of equine Neospora infection havebeen described. The parasites were identi®ed byimmunohistochemical labelling using N. caninumantibodies [59] and, in one case, serum antibodieswere also demonstrated by IFAT [60]. After iso-lation and characterisation of Neospora organ-isms from a horse in California, Marsh et al. [61]concluded that this parasite, primarily based onmolecular evidence, was distinct from N. cani-num, and they proposed the name Neosporahughesi. This species obviously has a high degreeof antigenic similarity with N. caninum and there-fore it is likely that serological assays based onN. caninum antigen would be useful for demon-stration of antibodies also in N. hughesi-infectedequines. A serological survey for Neospora infec-tion in horses was recently performed by theDAT [62].

6.5. Cat

So far, no naturally acquired cases of neos-porosis have been described in the cat. The IFATwas used in connection with experimental N.caninum infections [19, 63]. Cats inoculated orallywith N. caninum tissue cysts did not seroconvertas measured by the IFAT [64].

6.6. Rodents

Mice inoculated parenterally with N. caninumtachyzoites were shown by IFAT to develop anIgG response from day 9 after inoculation [65].The IFAT and iscom ELISA have beenemployed to screen murine hybridoma super-natants for mAbs to N. caninum [38, 66]. For thispurpose, IFAT has the advantage of allowing thevisual assessment of the localisation of the par-ticular mAbs on the parasite cell.


6.7. Primates

Neospora caninum has been shown to be trans-placentally transferred from experimentallyinoculated rhesus macaque dams to theirfoetuses [67]. Foetal IgG, IgM and IgA anti-bodies were demonstrated by IFAT. IscomELISA, IFAT and Western blot immunoassaywere used in a serological study on the possiblepresence of N. caninum in a population ofwomen with repeated abortion, but no serologi-cal evidence of human Neospora infection wasfound [68].

6.8. Birds

Natural Neospora infection has not beenobserved in birds. However, antibodies to N.caninum were demonstrated by IFAT and DATin blackbirds and starlings following experimen-tal infections [10].

7. Conclusion

The development of speci®c, sensitive and inex-pensive serological tests for N. caninum havebeen essential in the accumulation of knowledgeon the epidemiology of this important parasite.The development of serological assays for N.caninum antibodies corresponds generally to thatfor T. gondii during the previous decades. Withthe short time span since the discovery of N.caninum, an impressive number of serologicalmethods have so far been developed. Today,several tests are even commercially available.However, a problem with N. caninum serology isthat, at present, no ultimate, generally acceptedand standardised test is available. Most labora-tories involved in Neospora research and diagno-sis have developed their own assay procedures, aswell as cut-o� levels and other criteria for in-terpretation of results. Thus, it is not possible todirectly compare titres or absorbance valuesobtained in di�erent laboratories, even when thesame test has been used.

The antigen±antibody reactions producing ademonstrable test result are the summation of

several molecular interactions, and therefore min-ute changes in assay conditions can greatly a�ectthe test result. Also, each secondary antibody hasunique characteristics, such as a�nity for theparticular immunoglobulin antigen, that willa�ect titres or absorbance values. The lack ofstandardisation and the diversity of antigenpreparations and conjugates used in the di�erentN. caninum assays make it desirable with a criti-cal validation of the existing tests. To this end,there is an urgent need for well-characterised serafrom infected and non-infected animals for use inintercalibration studies. The establishment of in-ternational reference laboratories for Neosporadiagnosis should also be considered.

In some situations it is important to identifyan acute, acquired neosporosis, and to di�eren-tiate it from a chronic or persistent N. caninuminfection. Speci®c T. gondii antibodies generallyappear a week after acquisition of the infection.The IgM antibodies appear ®rst and arecommonly detectable for a few months.However, the individual variation is considerable,and speci®c IgM antibody levels can remain elev-ated, even for years [69]. In contrast to T. gondii,there is limited knowledge about the kinetics ofN. caninum IgM in di�erent hosts, and so farthere are no studies reported in dogs or cattle.Measurement of avidity of speci®c IgG anti-bodies has been used to diagnose recent primaryT. gondii infection in humans and to distinguishbetween primary and secondary infections [70].The N. caninum IgG avidity ELISA developedfor use with bovine serum needs large-scaleevaluation, but it appears to su�ciently discrimi-nate between recent and chronic N. caninuminfections in both experimental and naturallyinfected animals [41]. It would be worth evaluat-ing various N. caninum antigen preparations foruse in avidity ELISAs in order to optimise theprecision of the assay regarding its ability to datethe infection. Another approach to assess therecency of infection would be to identify and usestage-speci®c antigen preparations, includingsporozoite-, tachyzoite- and bradyzoite-speci®cantigens, respectively.

Neospora caninum serological assays are vitaltools with which to support results of clinical in-


vestigations, and they are indispensable in epide-miological studies. However, their optimal usefor diagnosis and research requires insight in dis-ease pathogenesis, epidemiology and statisticalinterference. With the diverse range of serologicaltests for N. caninum antibodies currently avail-able, each assay should be used critically and itsadvantages and drawbacks appreciated.

Acknowledgements

Drs Anna LundeÂ n and Peter Waller,SWEPAR, are thanked for valuable commentson the manuscript. The authors' research on N.caninum is supported by the Swedish Farmers'Foundation for Agricultural Research, theSwedish Council for Forestry and AgriculturalResearch, the Agria Research Fund and the Ivarand Elsa Sandberg Foundation.

References

[1] Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla

A. Newly recognized fatal protozoan disease of dogs. J

Am Vet Med Assoc 1988;192:1269±85.

[2] Holmdahl OJM, Mattsson JG, Uggla A, Johansson K-E.

The phylogeny of Neospora caninum and Toxplasma gon-

dii based on ribosomal RNA sequences. FEMS

Microbiol Lett 1994;119:187±92.

[3] Dubey JP, Lindsay DS. A review of Neospora caninum

and neosporosis. Vet Parasitol 1996;67:1±59.

[4] McAllister MM, Dubey JP, Lindsay DS, Jolley WR,

Wills RA, McGuire AM. Dogs are de®nitive hosts of

Neospora caninum. Int J Parasitol 1998;28:1473±8.

[5] BjerkaÊ s I, Presthus J. Immuno-histochemical and ultra-

structural characteristics of a cyst-forming sporozoon as-

sociated with encephalomyelitis and myosotis in dogs.

Acta Path Microbiol Scand 1988;96:445±54.

[6] Dubey JP. Recent advances in Neospora and neosporosis.

Vet Parasitol, 1999;84:349±367.

[7] Dubey JP, Hattel AL, Lindsay DS, Topper MJ.

Neonatal Neospora caninum infection in dogs: isolation

of the causative agent and experimental transmission. J

Am Vet Med Assoc 1988;193:1259±63.

[8] BjoÈ rkman C, LundeÂ n A, Holmdahl J, Barber J, Trees

AJ, Uggla A. Neospora caninum in dogs: detection of

antibodies by ELISA using an iscom antigen. Parasite

Immunol 1994;16:643±8.

[9] Romand S, Thulliez P, Dubey JP. Direct agglutination

test for serologic diagnosis of Neospora caninum infec-

tion. Parasitol Res 1998;84:50±3.

[10] Packham AE, Sverlow KW, Conrad PA et al. A modi-

®ed agglutination test for Neospora caninum: develop-

ment, optimization, and comparison to the indirect

¯uorescent-antibody test and enzyme-linked immunosor-

bent assay. Clin Diagn Lab Immunol 1998;5:467±73.

[11] Smith RD. Veterinary clinical epidemiology, 2nd ed.

Boca Raton: CRC Press, 1995.

[12] BjoÈ rkman C, Johansson O, Stenlund S, Holmdahl J,

Uggla A. Neospora species infection in a herd of dairy

cattle. J Am Vet Med Assoc 1996;208:1441±4.

[13] Barber JS, Trees AJ. Naturally occurring vertical trans-

mission of Neospora caninum in dogs. Int J Parasitol

1998;28:57±64.

[14] Sabin AB, Feldman HA. Dyes as microchemical indi-

cators of a new immunity phenomenon a�ecting a proto-

zoon parasite (Toxoplasma). Science 1948;108:660±3.

[15] Uggla A, Hilali M, LoÈ vgren K. Serological responses in

Sarcocystis cruzi infected calves challenged with

Toxoplasma gondii. Res Vet Sci 1987;43:127±9.

[16] PareÂ J, Hietala SK, Thurmond MC. Interpretation of an

indirect ¯uorescent antibody test for diagnosis of

Neospora sp. infection in cattle. J Vet Diagn Invest

1995;7:273±5.

[17] Hughes HPA. The antigenic structure of Toxoplasma

gondii. Lyon MeÂ d 1982;248:135±9.

[18] Trees AJ, Guy F, Tennant BJ, Balfour AH, Dubey JP.

Prevalence of antibodies to Neospora caninum in a popu-

lation of urban dogs in England. Vet Rec 1993;132:125±

6.

[19] Dubey JP, Lindsay DS, Adams DS et al. Serologic re-

sponses of cattle and other animals infected with

Neospora caninum. Am J Vet Res 1996;57:329±36.

[20] Fulton JD, Turk JL. Direct agglutination test for

Toxoplasma gondii. Lancet 1959;11:1068±9.

[21] Desmonts G, Remington JS. Direct agglutination test for

diagnosis of Toxoplasma infection: method for increasing

sensitivity and speci®city. J Clin Microbiol 1980;11:562±

8.

[22] Harlow E, Lane D. Immunoassay. In: Antibodies. A lab-

oratory manual. Cold Spring Harbor, NY: Cold Spring

Harbor Laboratory Press, 1988; 553±612.

[23] PareÂ J, Hietala SK, Thurmond MC. An enzyme-linked

immunosorbent assay (ELISA) for serological diagnosis

of Neospora sp. infection in cattle. J Vet Diagn Invest

1995;7:352±9.

[24] Huldt G. Workshop No. 3. Serodiagnosis of parasitic

infections. Parasitology 1981;82:49±55.

[25] Uggla A, Buxton D. Immune responses against

Toxoplasma and Sarcocystis infections in ruminants:

diagnosis and prospects for vaccination. Rev Sci Tech

O� Int Epizoot 1990;9:441±62.

[26] Williams DJL, McGarry J, Guy F, Barber J, Trees AJ.

Novel ELISA for detection of Neospora-speci®c anti-

bodies in cattle. Vet Rec 1997;140:328±31.


[27] LoÈ vgren K, Uggla A, Morein B. A new approach to the

preparation of a Toxoplasma gondii membrane antigen

for use in ELISA. J Vet Med B 1987;34:274±82.

[28] Lally NC, Jenkins MC, Dubey JP. Evaluation of two

Neospora caninum recombinant antigens for use in an

enzyme-linked immunosorbent assay for the diagnosis of

bovine neosporosis. Clin Diagn Lab Immunol

1996;3:275±9.

[29] Louie K, Swerlow KW, Barr BC, Anderson ML, Conrad

PC. Cloning and characterization of two recombinant

Neospora protein fragments and their use in serodiagno-

sis of bovine neosporosis. Clin Diagn Lab Immunol

1997;4:692±9.

[30] Dubey JP, Jenkins MC, Adams DS et al. Antibody re-

sponses of cows during an outbreak of neosporosis eval-

uated by indirect ¯uorescent antibody test and di�erent

enzyme linked immunosorbent assays. J Parasitol

1997;83:1063±9.

[31] BjoÈ rkman C, Holmdahl OJM, Uggla A. An indirect

enzyme-linked immunoassay (ELISA) for demonstration

of antibodies to Neospora caninum in serum and milk of

cattle. Vet Parasitol 1997;68:251±60.

[32] Baszler TV, Knowles DP, Dubey JP, Gay JM, Mathison

BA, McElwain TF. Serological diagnosis of bovine neos-

porosis by Neospora caninum monoclonal antibody-based

competitive inhibition enzyme-linked immunosorbent

assay. J Clin Microbiol 1996;34:1423±8.

[33] Wouda W, Brinkhof J, van Maanen C, de Gee ALW,

Moen AR. Serodiagnosis of neosporosis in individual

cows and dairy herds, a comparative study of three

enzyme-linked immunosorbent assays. Clin Diagn Lab

Immunol, in press.

[34] Osawa T, Wastling J, Maley S, Buxton D, Innes EA. A

multiple antigen ELISA to detect Neospora-speci®c anti-

bodies in bovine sera, bovine foetal ¯uids, ovine and

caprine sera. Vet Parasitol 1998;79:19±34.

[35] Moen AR, Wouda W, Mul MF, Graat EAM, van

Werven T. Increased risk of abortion following Neospora

caninum abortion outbreaks: a retrospective and prospec-

tive cohort study in four dairy herds. Theriogenology

1998;49:1301±9.

[36] BjoÈ rkman C, LundeÂ n A. Application of iscom antigen

preparations in ELISAs for diagnosis of Neospora and

Toxoplasma infections. Int J Parasitol 1998;28:187±93.

[37] LundeÂ n A, LoÈ vgren-Bengtsson K, SjoÈ lander A, Uggla A.

Iscoms in parasitological research. Parasitol Today

1996;12:320±3.

[38] BjoÈ rkman C, Hemphill A. Characterization of Neospora

caninum iscom antigens using monoclonal antibodies.

Parasite Immunol 1998;20:73±80.

[39] Huong LTT, LjungstroÈ m B-L, Uggla A, BjoÈ rkman C.

Prevalence of antibodies to Neospora caninum and

Toxoplasma gondii in cattle and water bu�aloes in

southern Vietnam. Vet Parasitol 1998;75:53±7.

[40] PareÂ J, Thurmond MC, Hietala SK. Neospora caninum

antibodies in cows during pregnancy as a predictor of

congenital infection and abortion. J Parasitol

1997;83:82±7.

[41] BjoÈ rkman C, NaÈ slund K, Stenlund S, Maley SW, Buxton

D, Uggla A. An IgG avidity ELISA to discriminate

between recent and chronic Neospora caninum infection.

J Vet Diagn Invest 1999;11:41±4.

[42] Jenkins MC, Wouda W, Dubey JP. Serological response

over time to recombinant Neospora caninum antigens in

cattle after a neosporosis-induced abortion. Clin Diagn

Lab Immunol 1997;4:270±4.

[43] Buxton D, Maley SW, Pastoret P-P, Brochier B, Innes

EA. Examination of red foxes (Vulpes vulpes) from

Belgium for antibody to Neospora caninum and

Toxoplasma gondii. Vet Rec 1997;141:308±9.

[44] Barber JS, Gasser RB, Ellis J, Reichel MP, McMillan D,

Trees AJ. Prevalence of antibodies to Neospora caninum

in di�erent canid populations. J Parasitol 1997;83:1056±

8.

[45] Conrad PA, Sverlow K, Anderson M et al. Detection of

serum antibody responses in cattle with natural or exper-

imental Neospora infections. J Vet Diagn Invest

1993;5:572±8.

[46] Gottstein B, Hentrich B, Wyss R et al. Molecular and

immunodiagnostic investigations on bovine neosporosis

in Switzerland. Int J Parasitol 1998;28:679±91.

[47] Thurmond MC, Hietala SK. E�ect of congenitally

acquired Neospora caninum infection on risk of abortion

and subsequent abortions in dairy cattle. Am J Vet Res

1997;58:1381±5.

[48] Dannatt L. Neospora caninum antibody levels in an ende-

mically-infected dairy herd. Cattle Pract 1997;5:335±7.

[49] Dubey JP, Romand S, Hilali M, Kwok OCH, Thulliez P.

Seroprevalence of antibodies to Neospora caninum and

Toxoplasma gondii in water bu�aloes (Bubalus bubalis)

from Egypt. Int J Parasitol 1998;28:527±9.

[50] Schares G, Peters M, Wurm W, BaÈ rwald A, Conraths

FJ. The e�ciency of vertical transmission of Neospora

caninum in dairy cattle analysed by serological tech-

niques. Vet Parasitol 1998;80:87±98.

[51] Barr BC, Anderson ML, Sverlow KW, Conrad PA.

Diagnosis of bovine fetal Neospora infection with an

indirect ¯uorescent antibody test. Vet Rec 1995;137:611±

3.

[52] Wouda W, Dubey JP, Jenkins MC. Serological diagnosis

of bovine fetal neosporosis. J Parasitol 1997;83:545±7.

[53] Otter A, Je�rey M, Scholes SFE, Helmick B, Wilesmith

JW, Trees AJ. Comparison of histology with maternal

and fetal serology for the diagnosis of abortion due to

bovine neosporosis. Vet Rec 1997;141:487±9.

[54] Dubey JP, Hartley WJ, Lindsay DS, Topper MJ. Fatal

congenital Neospora caninum infection in a lamb. J

Parasitol 1990;76:127±30.

[55] McAllister MM, McGuire AM, Jolley WR, Lindsay DS,

Trees AJ, Stobart RH. Experimental neosporosis in preg-

nant ewes and their o�spring. Vet Pathol 1996;33:647±

55.


[56] Buxton D, Maley SW, Thomson KM, Trees AJ, Innes

EA. Experimental infection of non-pregnant and preg-

nant sheep with Neospora caninum. J Comp Pathol

1997;117:1±16.

[57] Dubey JP, Morales JA, Villalobos P, Lindsay DS,

Blagburn BL, Topper MJ. Neosporosis-associated abor-

tion in a dairy goat. J Am Vet Med Assoc 1996;208:263±

5.

[58] Engeland IV, Waldeland H, Andresen é, Lùken T,

BjoÈ rkman C, BjerkaÊ s I. Foetal loss in dairy goats: an epi-

demiological study in 22 herds. Small Rum Res

1998;30:37±48.

[59] Dubey JP, Porter®eld ML. Neospora caninum

(Apicomplexa) in an aborted equine fetus. J Parasitol

1990;76:732±4.

[60] Daft BM, Barr BC, Collins N, Sverlow K. Neospora

encephalomyelitis and polyradiculoneuritis in an aged

mare with Cushing's disease. Eq Vet J 1996;28:240±3.

[61] Marsh AE, Barr BC, Packham AE, Conrad PA.

Description of a new Neospora species (Protozoa:

Apicomplexa: Sarcocystidae). J Parasitol 1998;84:983±91.

[62] Dubey JP, Romand S, Thulliez P, Kwok OCH, Shen

SK, Gamble HR. Prevalence of antibodies to Neospora

caninum in horses in North America. J Parasitol 1999, in

press.

[63] Dubey JP, Lindsay DS. Transplacental Neospora cani-

num infection in cats. J Parasitol 1989;75:765±71.

[64] McAllister MM, Jolley WR, Wills RA, Lindsay DS,

McGuire AM, Tranas JD. Oral inoculation of cats with

tissue cysts of Neospora caninum. Am J Vet Res

1998;59:441±4.

[65] Lindsay DS, Dubey JP. Neospora caninum (Protozoa:

Apicomplexa) infections in mice. J Parasitol 1989;75:772±

9.

[66] Cole RA, Lindsay DS, Dubey JP, Blagburn BL.

Detection of Neospora caninum in tissue sections using a

murine monoclonal antibody. J Vet Diagn Invest

1993;5:579±84.

[67] Barr BC, Conrad PA, Sverlow KW, Tarantal AF,

Hendrickx AG. Experimental fetal and transplacental

Neospora infection in the nonhuman primate. Lab Invest

1994;71:236±42.

[68] Petersen E, Lebech M, Jensen L et al. Neospora caninum

infection and repeated abortion in humans. Emerg Infect

Dis 1999;5:278±80.

[69] Ho-Yen DO. Immunocompromised patients. In: Ho-Yen

DO, Joss AWL, editors. Human toxoplasmosis. Oxford:

Oxford University Press, 1992;185±203.

[70] Jenum PA, Stray-Pedersen B, Gundersen A-G. Improved

diagnosis of primary Toxoplasma gondii infection in early

pregnancy by determination of antitoxoplasma immuno-

globulin G avidity. J Clin Microbiol 1997;35:1972±7.


Documents

Serological diagnosis of Neospora caninum infection