JOURNAL OF CLINICAL MICROBIOLOGY, JUly 1994, p. 1674-1679 Vol. 32, No. 70095-1 137/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Competitive Inhibition Enzyme-Linked Immunosorbent Assay forAntibody in Sheep and Other Ruminants to a Conserved

Epitope of Malignant Catarrhal Fever VirusHONG LI,1'2 DAVID T. SHEN,2 DONALD P. KNOWLES,2 JOHN R. GORHAM,2

AND TIMOTHY B. CRAWFORD'*Department of Veterinary Microbiology and Pathology, Washington State University,1 and Animal Diseases

Research Unit, USDA Agyicultural Research Service,2 Pullman, Washington 99164

Received 11 March 1994/Accepted 12 April 1994

Malignant catarrhal fever (MCF) is a severe, usually fatal, acute systemic disease syndrome of certaindomestic and wild ruminants caused by members of the family Gammaherpesvirinae. Two distinct but closelyrelated viruses cause clinically indistinguishable syndromes: one that is indigenous to the wildebeest and theother that apparently is indigenous to domestic sheep. Neither the pathogenesis nor the epidemiology ofsheep-associated MCF (SA-MCF) is understood, primarily because of a lack of adequate detection methods forthe etiologic agent or antibody against it. No acceptably documented isolates of SA-MCF virus have beenreported, and existing antibody assays suffer from significant cross-reactivity with other viruses. As a basis fora specific serologic assay, an attempt was made to identify an epitope conserved among all isolates of MCFviruses, by using a monoclonal antibody (MAb) produced against a previously reported U.S. isolate of MCFvirus. A MAb (15-A) which bound a conserved epitope present on all four isolates of MCF virus examined wasfound. MAb 15-A did not react with eight common sheep and goat viruses or five common bovine viruses.Immunoprecipitation revealed that the 15-A epitope was located on the viral glycoprotein complex, withmolecular masses of 115, 110, 105, 78, and 45 kDa. Sera from experimentally and naturally infected animalswhich yielded a similar glycoprotein complex immunoprecipitation pattern competed with MAb 15-A for itsepitope. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on MAb 15-A wastherefore developed. The assay detected antibody in inapparently infected sheep and in cattle, deer, and bisonwith clinical MCF. Of the 149 serum samples from sheep associated with MCF outbreaks, 88 (55%) wereseropositive by competitive inhibition ELISA.

Malignant catarrhal fever (MCF) is a frequently fatal lym-phoproliferative and inflammatory disease syndrome of certaindomestic and wild ruminant species caused by closely relatedmembers of the family Gammaherpesvirinae (3). Two majorepidemiologic forms of MCF, defined by the reservoir rumi-nant species from which the causative virus arises, are recog-nized. One, known as the African form, is referred to aswildebeest-associated MCF (4, 32, 33). The etiology is arelatively well-characterized lymphotrophic gammaherpesvi-rus, named alcelaphine herpesvirus 1 (AHV-1) (37), which isindigenous in the wildebeest (subfamily Alcelaphinae) to whichit is well adapted and nonpathogenic.The other major, worldwide source of an MCF agent is

thought to be domestic sheep. Though never successfullypropagated in vitro, there is substantial epidemiologic andserologic evidence that a virus closely related to AHV-1 existsin sheep and is transmitted, often at lambing, to susceptible,poorly adapted ruminants such as domestic cattle, deer, andbison (29, 31, 35). Sheep-associated malignant catarrhal fever(SA-MCF) is one of the few known infectious diseases forwhich an etiologic agent has never been isolated. Moreover,experimental transmission of MCF to cattle by inoculation ofmaterial from suspect sheep has been almost uniformly unsuc-cessful (8, 15). However, recent demonstration of SA-MCFvirus (SA-MCFV) DNA sequences with homology to Epstein-Barr virus and herpesvirus saimiri in leukocytes from normal

* Corresponding author. Mailing address: Department of Veteri-nary Microbiology and Pathology, Washington State University, Pull-man, WA 99164.

sheep and SA-MCF-affected cattle (2) strengthens the notionthat sheep are indeed carriers of a gammaherpesvirus. So far,productive replication in vitro from infected sheep leukocyteshas not been achieved, which suggests that sheep cells may notbe highly permissive. Restricted gene expression in lympho-cytes is a common feature of the gammaherpesviruses, e.g.,Epstein-Barr virus (18), herpesvirus saimiri (9), and Marek'sdisease virus (40).MCF usually is sporadic but occasionally occurs epizooti-

cally, causing significant losses (5, 28, 30, 36). The lack ofavailable confirmed sheep-associated isolates has hindereddevelopment of efficient reagents for diagnosis and control ofSA-MCF. The identification of MCFV-infected sheep cur-rently is based on the detection of antibodies to AHV-1, thewildebeest isolate (14, 26, 41). Only low titers of neutralizingantibody to AHV-1 are found in infected sheep (11, 38),limiting the utility of the neutralization test. Indirect immuno-fluorescence (IIF) and enzyme-linked immunosorbent assay(ELISA), using AHV-1-infected cells as antigens, are the testscurrently in most common use (14, 38, 41). Their accuracy isalso limited, however, by the fact that MCFVs share antigenswith other bovine herpesviruses (13, 23). Thus, insensitivityand nonspecificity seriously limit the usefulness of the cur-rently available tests for studying the epidemiology, pathogen-esis, and control of SA-MCF (11, 12, 22). We report here a testfor MCFV antibody, based on a single epitope shared by allMCFV isolates but no other ruminant viruses that we haveexamined to date, and describe its use in the detection ofMCFV antibody in sheep and other ruminant species.

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DETECTION OF MCFV ANTIBODY BY CI-ELISA 1675

MATERLILS AND METHODS

Viruses and viral antigens. The Minnesota isolate ofMCFV, originally derived from a cow involved in a sheep-associated MCF outbreak in Minnesota (10) (herein desig-nated MN-MCFV), WC-11 (cell culture passaged strain ofAHV-1), and fetal mouflon sheep kidney (FMSK) cells werekindly provided by W. Heuschele, Center for Reproduction ofEndangered Species, Zoological Society of San Diego, SanDiego, Calif. Bovine herpesvirus 1 (BHV-1) (LA strain, ATCCVR-188), BHV-4 (DN-599, ATCC VR-631), and bovine tur-binate cells (ATCC CRL-1390) were obtained from the Amer-ican Type Culture Collection (Rockville, Md.). Bovine viraldiarrhea virus, parainfluenza virus 3, ovine respiratory syncytialvirus, bovine respiratory syncytial virus, ovine adenoviruses5and 6, ovine herpesvirus 1, and caprine herpesvirus 1 werekindly supplied by J. Evermann, Washington Animal DiseaseDiagnostic Laboratory, Pullman. The MN-MCFV was propa-gated on FMSK cells at the 16th to 22nd passage level in highglucose Dulbecco's modified Eagle medium (DMEM) contain-ing 10% fetal bovine serum. All other viruses were produced inbovine turbinate cells grown in DMEM with 10% fetal bovineserum. MCFV antigens were prepared by infection of FMSKcell monolayers in 900-cm2 roller bottles at a multiplicity ofinfection of 0.1. When 80 to 90% of the cells exhibitedcytopathic effect, the supernatant was harvested and clarifiedby centrifugation at 4,300 x g for 30 min, and virus waspelleted at 125,000x g for 90 min through a 2-cm 35% (wt/wt)sucrose column. The pellets were resuspended in phosphate-buffered saline (PBS), sonicated, and stored at -20°C.

Sera. During 1992 and 1993, serum samples were obtainedfrom sheep, cattle, and deer in seven states that had beenassociated with outbreaks of MCF (see Table 2), diagnoseshaving been established by clinical signs and pathologic lesions.MCFV antibody-negative sheep sera were obtained from 5- to6-month-old lambs in a local farm flock in Pullman, Wash.Antisera to MN-MCFV were raised in rabbits by immunizationwith MN-MCFV antigen prepared as described above. Rabbitantiserum against the WC-11 strain of AHV-1 was purchasedfrom a commercial source (Cytimmune, San Diego, Calif.).Sera from naturally infected wildebeest and other exoticruminants were supplied by W. Heuschele. Sera from sheepexperimentally inoculated with a U.S. isolate of AHV-1 (4, 41)were kindly provided by A. Castro, Pennsylvania State Univer-sity. Bovine antisera to MCFV 732, a putative SA-MCFVisolate from Austria (39), and to BHV-1, BHV-2, BHV-4, andbovine viral diarrhea virus were kindly provided by J. Pearson,National Veterinary Service Laboratory, USDA Animal andPlant Health Inspection Service, Ames, Iowa. Antisera toCAEV and OPPV were obtained from the Washington AnimalDisease Diagnostic Laboratory, Pullman. Antisera to ovineherpesvirus 1 and caprine herpesvirus were raised in mice byinoculation of virus propagated on sheep kidney cells andpurified by sucrose gradient centrifugation as described above.

Radioimmunoprecipitation. Radioimmunoprecipitation oflabeled viral proteins was performed as previously described(20). Briefly, monolayers of FMSK cells were infected withMN-MCFV at a multiplicity of infection of 3.0. [35S]methi-onine or [3H]glucosamine (40 pRCi/ml; New England Nuclear,Boston, Mass.) was added to the medium, and the cultureswere harvested at 90% cytopathic effect. For radioimmunopre-cipitation, labeled cell lysates were mixed with polyclonalantisera or monoclonal antibodies (MAbs), and the antigen-antibody complexes were allowed to bind to recombinantprotein G-coated beads (GENEX Co., Gaithersburg, Md.).The beads were then precipitated and washed, bound com-

plexes were solubilized, and the solubilates were subjected tosodium dodecyl sulfate-polyacrylamide gel electrophoresis.

IIF assay. FMSK cells infected at a multiplicity of infectionof 0.5 with MN-MCFV were harvested at 24 h postinfection.Bovine turbinate cells infected with other viruses were har-vested at 50% cytopathic effect. Harvested cells were washedwith PBS, dried onto glass slides, and fixed with 100% acetoneat 25°C for 5 min. Thirty microliters of twofold dilutions ofantisera or MAbs was layered onto the cells and incubated at37°C for 1 h. After the cells were washed with PBS, 30 [lI of apredetermined optimal dilution of commercial recombinantprotein G conjugated with fluorescein isothiocyanate (ZymedLab, Inc., San Francisco, Calif.) was added to each well andincubated at 37°C for1 h. Uninfected control cells were stainedin the same manner.CI-ELISA. A panel of MAbs to MN-MCFV was produced

and characterized as described elsewhere (21). MAbs with highactivity in ELISA were evaluated for their ability to competefor their epitopes with antibodies from sheep seropositive forMCFV as defined byIIF and radioimmunoprecipitation. Com-petitive inhibition ELISA (CI-ELISA) was performed by usinga modification of a previously described procedure (1, 19).Ninety-six-well polystyrene plates (Immulon 4; Dynatech Lab,Inc., Chantilly, Va.) were coated at 4°C for 18 to 20 h with 0.25,ug of the above-described MCFV antigen in 50 mM carbon-ate-bicarbonate buffer (pH 9.6) per well. Control wells werecoated with the proteins from uninfected FMSK culturessubjected to the same purification procedure. After the wellswere blocked with 20% nonfat milk in PBS at 25°C for 2 h andwashed with PBS with 0.1% Tween 20, 250 plI of dilutions oftest serum and 50 ,lI of MAb (0.2 ,ug) were added andincubated at 25°C for 1 h. The wells were washed three timeswith PBS-Tween 20 and incubated with alkaline phosphataseanti-mouse immunoglobulin G (Sigma, St. Louis, Mo.) at 25°Cfor 1 h. After a final wash, 50 pLI of substrate buffer containing1 mg of p-nitrophenyl phosphate (Sigma) per ml was added,the mixture was incubated, and the optical density at 414(OD414) was determined. Serum samples were run in triplicate.Negative control sera were defined by an absence of MCFVantibody detectable by IIF and immunoprecipitation. Whenthe mean of the three replicate OD readings of a serum wasmore than 3 standard deviations below the mean of a panel ofsix negative control serum samples, run in triplicate, the serumwas considered positive for MCFV antibody.

RESULTS

Identification of an epitope conserved among isolates ofMCFV. Sixty-four MAbs with high reactivity in ELISA wereselected and tested against sheep sera by CI-ELISA. MAb15-A was inhibited by sera from sheep that tested positive forMCFV antibody in IIF and immunoprecipitation. MAb 15-Aof isotype immunoglobulin G2b, when used in immunoprecipi-tation, bound an epitope located on a glycoprotein complexwhich, on reducing gels, resolved into five bands with apparentmolecular masses of 115, 110, 105, 78, and 45 kDa (Fig. 1A).MAb 15-A identified only a 45-kDa protein in immunoblots(Fig. 1B).

Figure 2 shows viral proteins of MN-MCFV which reacted inimmunoprecipitation with sera from wildebeest, sheep, cattle,and deer experimentally or naturally infected with a variety ofisolates of MCFV. The predominant proteins recognized bythese sera were a single 130-kDa protein; a complex of proteinswith molecular masses of 115, 110, 105, 78, and 45 kDa; and asingle 110-kDa protein which comigrated with the 110-kDamoiety of the complex. To identify a conserved epitope, sera

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A B1 2 3

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FIG. 1. (A) Immunoprecipitation of [35S]methionine-labeled pro-teins of MN-MCFV-infected (lanes V) or uninfected (lanes C) celllysate with MAb 15-A (lanes labeled 1) and isotype MAb control(lanes labeled 2). (B) Immunoblotting of purified MN-MCFV (lanesV) and FMSK cell (lanes C) antigens with MAb 15-A (lanes labeled 3)and isotype MAb control (lanes labeled 4). Molecular mass markers(in thousands) are indicated on the left.

from the animals listed above that were positive for MCFV byimmunoprecipitation were examined for their ability to blockthe binding of MAb 15-A in CI-ELISA. MAb 15-A wasefficiently inhibited by antisera against a variety of MCFVisolates of both wildebeest and sheep origin but was notsignificantly inhibited by the preinoculation sera from the sameanimals or from other uninoculated control animals of thesame species (Fig. 3).

Specificity of MAb 15-A for MCFV. To provide evidence ofthe specificity of MAb 15-A, rabbits were immunized byintramuscular injection with purified MN-MCFV with Titer-Max adjuvant (Vaxcel, Inc., Norcross, Ga.) and tested forantibody by CI-ELISA. Sera from these rabbits competedeffectively with MAb 15-A in CI-ELISA, in contrast to serafrom the rabbit immunized with uninfected FMSK cell antigen(Fig. 4). Moreover, sera from rabbits immunized with the

1 2 3 4 5

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29

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FIG. 2. Immunoprecipitation of [35S]methionine-labeled proteinsof MN-MCFV-infected (lanes V) or uninfected (lanes C) cell lysatewith antisera (lanes labeled +) and preinoculated (lanes labeled -) ornegative control sera (-*) from the following animals inoculated or

infected with different MCFV isolates: a rabbit immunized withAHV-1 (strain WC-11) (lanes 1), a rabbit immunized with MN-MCFV(lanes 2), a calf inoculated with a putative SA-MCFV isolate fromAustria (MCFV 732) (lanes 3), sheep experimentally inoculated with a

U.S. isolate of AHV-1 (lanes 4), wildebeest naturally infected withMCFV (lanes 5), deer with clinical SA-MCF (lanes 6), and sheepnaturally infected with MCFV (lanes 7). Molecular mass markers (inthousands) are indicated on the left.

Ra Sh Bo Dr Wb

FIG. 3. The conservation of 15-A epitope among MCFV isolates.Expressed as the percentage of inhibition in CI-ELISA by sera from a

rabbit immunized with AHV-1 (strain WC-1 1) (Ra), sheep experimen-tally inoculated with a U.S. isolate of AHV-1 (Sh), a calf inoculatedwith a putative SA-MCFV isolate from Austria (MCFV 732) (Bo),deer with clinical SA-MCF (Dr), and wildebeest naturally infected withMCFV (Wb). See Materials and Methods for serum sources.

WC-11 strain of AHV-1, from sheep inoculated with a U.S.isolate of AHV-1 (4, 43), and a calf infected with a putativeSA-MCFV isolate (MCFV 732) from Austria, which hasrecently been reported (39), strongly competed with the MAb15-A in CI-ELISA (Fig. 3). When tested by IIF and CI-ELISA,MAb 15-A did not react with any of eight common sheep andgoat viruses or five common bovine viruses (Table 1).

Detection ofMCFV antibody in sheep and other ruminants.Sera from 12 sheep that were seronegative by IIF and immu-noprecipitation yielded an OD value of 0.8 ± 0.05 (mean ±

standard deviation) by CI-ELISA, representing approximately20% inhibition of the OD of MAb 15-A alone. The mean ODof these 12 negative serum samples, as well as the MAb isotypecontrol of irrelevant specificity, yielded an OD value of .0.1.Comparison of both CI-ELISA ODs and immunoprecipitationpatterns of seronegative lambs 5 to 6 months of age and 36seronegative adult sheep revealed no significant differencesbetween the two age groups. Of 149 serum samples collectedfrom sheep associated with MCF outbreaks in seven states, 88

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80 -

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FIG. 4. Specificity of MAb 15-A in CI-ELISA revealed by antiserafrom rabbits immunized with MN-MCFV antigens (A and B) or FMSKcell antigens (C).

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TABLE 1. Reactivity of MAb 15-A with ovine, caprine,and bovine viruses

IIF reactivity to:Virus Immune MAb CI-ELISA

serumb 15-AC

Ovine herpesvirus 1 + - NDdCaprine herpesvirus 1 + - NDOPPV + - -CAEV + - -

Ovine adenovirus 5 + - -

Ovine adenovirus 6 + - -

Ovine respiratory syncytial virus + - NDOvine parainfluenza virus 3 + - -BHV-1 + - -BHV-2 ND - -BHV-4 + - -Bovine viral diarrhea virus + - -

Bovine respiratory syncytial virus + - -

MN-MCFV + + +MCFV 732e + + +AHV-1 strain WC-11 + + +

a Sera negative for MCFV had less than 35% inhibition of MAb 15-A.b The IIF titers of all antisera exceeded 1:500.c MAb 15-A negative at 40 ,ug/ml; MAb 15-A positive at 0.002 ,ug/ml.d ND, not done.e Putative SA-MCFV from Austria.

were positive by CI-ELISA, inhibiting the test by >3 standarddeviations more than the mean inhibition value for the nega-tive controls. All sera positive by CI-ELISA were also positiveby IIF. The converse, however, was not true: some serumsamples that were positive by IIF were negative by CI-ELISA(Table 2). Of 65 lambs tested that were less than 1 year old,only 2 were seropositive by CI-ELISA, whereas a high percent-age of sheep over 1 year old were seropositive (Table 3). Thetest detected antibody in six of eight cows, two of three deer,and two bison with clinical MCF.The sensitivity of CI-ELISA was comparable to that of IIF.

Antibody titers of sera from sheep experimentally infected witha U.S. isolate of AHV-1 (4, 41) as measured by the two assayswere not significantly different (Fig. 5). Moreover, when sam-ples were compared at only a single dilution, the percentageinhibition in CI-ELISA was generally proportional to theimmunofluorescent staining intensity (data not shown). Care-ful optimization of the coating antigen concentration signifi-cantly improved the sensitivity of the assay (data not shown).The concentration of the MAb, however, was less critical,suggesting that it might have a low binding affinity (16).

TABLE 2. Seroprevalence of MCFV antibody in sheep associatedwith MCF outbreaks

Species clinically Location of No. of No. positive by:affected outbreak sheep tested CI-ELISA IF

Cattle California 15 10 14Cattle Georgia 13 1 1Cattle North Dakota 14 14 14Cattle Wyoming 48 19 27Deer North Dakota 7 5 6Deer Tennessee 11 11 11Deer Washington 21 11 15Bison Kentucky 20 18 18

Total 149 88 106

TABLE 3. The relationship between age of sheep and presence ofMCFV antibody by CI-ELISA

Age (yr) No. tested No. positive % Positive

<1 65 2 31-3 57 46 81>3 49 45 92

DISCUSSION

MCF is a very complex syndrome that, in spite of manystudies over more than 50 years, is only superficially under-stood. The sheep-associated form, in particular, has eludedefforts to define its etiology, epidemiology, and pathogenesis.Though considerable evidence has accumulated implicatingdomestic sheep as the reservoir, no confirmed isolate yet existsand Koch's postulates have not been fulfilled. Its usuallysporadic nature, obscure transmission patterns, and lack ofspecific diagnostic tests for inapparent carriers have hindereddevelopment of control measures for the disease. The designof this study was to identify an epitope specific for MCFV andconserved among all of its known isolates, of both sheep andwildebeest origin, and develop a seroassay based on thespecificity of a MAb for that epitope.The data of this report characterize a murine immunoglob-

ulin G2b (MAb 15-A) that defines a conserved carbohydrate-dependent epitope on MCFV. This epitope was present in allisolates of MCFV tested but was not present in 13 commonsheep and cattle viruses.

Parallel testing of 149 serum samples from sheep that hadbeen in contact with cattle, deer, and bison during MCFoutbreaks revealed 83% concordance between CI-ELISA andIIF. The number of seropositive sheep detected by IIF wasalways equal to or greater than the number detected byCI-ELISA. Sharing of antigens between MCFV and otherruminant herpesviruses, such as BHV-1, BHV-2, and BHV-4and goat herpesvirus 1, is well-known (13, 23). Cross-reactivitybetween MCFV and viruses such as these apparently is respon-sible for the greater number of sheep scored seropositive byIIF than by CI-ELISA. Another source of nonspecificitypotentially affecting an immunofluorescence assay is binding ofantibodies to Fc receptors on herpesvirus-infected cells, as has

100

i:=a>_ *~~~~~~~~sheep A

20 40 80 1 60 320 640

Reciprocal of Dilutions

FIG. 5. Comparison of sensitivities of CI-ELISA and IIF (inset) indetection of specific antibody in sera from sheep experimentallyinoculated with a U.S. isolate of AHV-1 (A and B) and control (C).

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been shown with herpes simplex virus (42, 43) and humancytomegalovirus (17, 34).Two hundred forty-seven of 555 (44.55%) sheep serum

samples collected from seven different states were positive byCI-ELISA. One hundred forty-nine of the 555 samples werefrom sheep known to have been associated with MCF out-breaks in cattle, deer, or bison. Eighty-eight of these 149 serumsamples (55%) were positive. This relatively high percentage ofseropositivity in sheep is consistent with earlier studies basedon IIF and indirect ELISA, in which infection rates rangingfrom 22 to 97% were reported (22, 23, 38, 41). The wide rangemay reflect differences in the ages of sheep examined, as wellas the relatively low specificities of these assays. In the presentstudy, a very low rate of seropositivity was present for lambsunder 1 year of age. It was much higher for sheep 2 years of ageand older. A better definition of the relationship betweenseropositivity and age requires examination of a larger set ofsequentially collected samples, which is underway.The pattern suggested by the data herein, perhaps surpris-

ingly, contrasts with that which is usually seen in wildebeestcalves infected with AHV-1, apparently at birth (25, 31). Theinfected calves shed virus into the environment, serving as animportant source of disease transmission (27). Neutralizingantibody to AHV-1 is usually detectable in wildebeest calves by3 months of age (24), after which shedding ceases. Theserological pattern in sheep suggested by this study is reminis-cent, however, of that of natural infections of squirrel monkeyswith herpesvirus saimiri, another gammaherpesvirus, whereinthe young are infected not at birth but sometime after 12months of age. By 2 years of age, virtually all young squirrelmonkeys born of persistently infected mothers have serocon-verted (6, 7). Whether the pattern in sheep reflects delayedinfection or a delayed immune response to an earlier infectionremains to be determined.Only limited numbers of serum samples were examined

from other ruminant species, both domestic and wild. Aseroprevalence rate generally resembling that found in domes-tic sheep was found in domestic goats and bighorn sheep. Incontrast, other ruminant species, such as llamas and mountaingoats, were found not to exhibit significant rates of seroposi-tivity (data not shown). Preliminary studies have found signif-icant concordance between CI-ELISA and detection of theagent by PCR, using primers derived from a recently reportedsequence of an SA-MCFV-specific DNA fragment (2). Studiesto better define the epidemiologic patterns of this agent invarious wild and domestic ruminant species are currentlyongoing.

ACKNOWLEDGMENTS

We thank Donal O'Toole, John Vander-Schealie, and Lee Bates forvaluable serum samples from sheep and cattle with clinical MCF andDongyue Zhuang and Soonhee Kwon for excellent technical assis-tance. Appreciation is also expressed to Travis McGuire, DouglasJasmer, and William Davis for valuable discussions and suggestions.

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