characterization of strains of mycoides small colony type … · contagious bovine pleuropneumonia...

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Apr. 2000, p. 1419–1425 Vol. 38, No. 4

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Characterization of Strains of Mycoplasma mycoides subsp. mycoidesSmall Colony Type Isolated from Recent Outbreaks of Contagious

Bovine Pleuropneumonia in Botswana and Tanzania:Evidence for a New Biotype

JOHN B. MARCH,* JASON CLARK, AND MALCOLM BRODLIE

Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, Scotland, United Kingdom

Received 25 August 1999/Accepted 24 January 2000

Four strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated from recentoutbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswananstrain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolatesand older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differ-ences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSCrabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertionsequence analysis using IS1634 clearly indicates a close evolutionary relationship to west African strains,hybridization with IS1296 shows the absence of a band present in all other strains of MmmSC examined. Thedata suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, includingpoor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are alsoexhibited by Mycoplasma capricolum subsp. capripneumoniae (causal agent of contagious caprine pleuropneu-monia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the variousMmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to belung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case ofCBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/ora potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturallyinfected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not againstany other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a setof protective antigens different from those of other strains of MmmSC (including vaccine strains). Thesefindings have implications for the control of the current CBPP epidemic in Africa.

Contagious bovine pleuropneumonia (CBPP), caused by in-fection with Mycoplasma mycoides subsp. mycoides small col-ony biotype (MmmSC), is one of the major infectious diseasesaffecting cattle in Africa. CBPP has spread alarmingly duringthe 1990s, infecting several countries previously free from thedisease, and was recently reported by the Office Internationaldes Epizooties as causing greater losses in cattle than any otherdisease, including rinderpest (27). Current losses are estimatedto be in the region of $2 billion per annum (23). Contributoryfactors to this current resurgence are thought to include thebreakdown of veterinary services (30), increased and unre-stricted cattle movements (due to drought, war, and civil strife(38), and a lack of vaccine efficacy (cited in references 24 and36). This paper investigates the additional possibility that anew biotype of the causal organism may be involved in currentoutbreaks.

Studies to date on the molecular epidemiology of MmmSCare consistent with there being two main subtypes: those iso-lated from recent European outbreaks (post-1980) and thoseisolated from African and Australian outbreaks, some of whichdate back to 1936 (e.g., the Australian vaccine strain V5 [6]).This classification is based on a variety of criteria (for reviewsee reference 21). Insertion sequence IS1296 banding patterns

(3) and restriction fragment length polymorphisms (RFLPs)(29) both suggest that European isolates fall into a singlehomogenous group, while the African and Australian strainsform a separate, more heterogeneous grouping. This hetero-geneity is most likely due to the widely separated geographicaland temporal origins of the African and Australian strainsstudied (1931 to 1993), in contrast to those from Europeanoutbreaks (post-1980), which have been collected over a rela-tively short period of time. Similarly, antigenic profiling ofEuropean and African and Australian strains has revealedconsistent differences between the two types (for example thepresence of a 70- to 72-kDa band in African and Australianstrains which is absent from European isolates, among otherdifferences (8, 9, 29). Biochemical analyses (1, 12, 13) have alsorevealed a systematic difference between European and Afri-can and Australian strains, as the latter group are able tooxidize glycerol at high rates in vitro.

RFLP analysis of recent African field isolates has revealedsome variation in the patterns observed between differentstrains (36). Although the importance of these findings onvirulence and pathogenicity is not known, they do raise thepotential that antigenic drift may have occurred among newerfield isolates (which could affect current vaccine efficacy). Wehave conducted a detailed investigation of four strains ofMmmSC isolated from the recent outbreaks of CBPP in Bo-tswana and Tanzania. Prior to these outbreaks both countrieshad been free from the disease for many years (46 and 25years, respectively [5]). Thus, the isolates should represent

* Corresponding author. Mailing address: Moredun Research Insti-tute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scot-land, United Kingdom. Phone: 44 131 445 5111. Fax: 44 131 445 6235.E-mail: [email protected].

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new, virulent outbreaks of CBPP rather than older endemictypes. Findings suggest that while three recent isolates arehighly similar to each other (and also display similarities toolder strains of MmmSC), a fourth strain, isolated from Bo-tswana in 1995, exhibits considerable phenotypic differences.These differences may have both evolutionary and disease con-trol implications.

MATERIALS AND METHODS

Mycoplasma strains and growth conditions. M. mycoides subsp. mycoides SCstrains used in this study are shown in Table 1. Current field strains (N6, M375,Tan1, and Tan8) were clonally isolated three times, and their identity as MmmSCwas confirmed by PCR (15). Mycoplasma capricolum subsp. capripneumoniaestrain 19/2 (16) was obtained from Gareth Jones at Moredun Research Institute,Penicuik, United Kingdom. Unless otherwise stated, MmmSC strains weregrown in Gourlay’s medium containing 20% heat-inactivated horse or pig serum(broth or agar) (10) containing 0.25 mg of ampicillin/ml and 0.025% thallousacetate at 37°C in a 5% CO2 atmosphere. Mycoplasma Experience (ME;Reigate, Surrey, United Kingdom) culture medium (broth and agar) was used toculture M. capricolum subsp. capripneumoniae under identical conditions.

Growth curves. A 1-ml aliquot of frozen culture (approximately 108 CFU) wasremoved from 280°C storage, thawed for 1 h at room temperature, and trans-ferred into 10 ml of fresh Gourlay’s broth (GB) prewarmed to 37°C. The culturewas then incubated for a minimum of 16 h at 37°C to ensure that bacteria werein the logarithmic phase of growth. Every 2 h thereafter, 0.1-ml samples wereremoved for estimation of viable counts by dilution into fresh GB in the range1021 to 1027 and by plating 0.25 ml onto prewarmed Gourlay’s plates. Platescontaining between approximately 10 and 1,000 colonies were counted with theaid of a hand-operated counter to estimate the titer at the time of sampling.Growth curves were performed on three separate occasions for each strain, withthe exception of Gladysdale (five times) and 6479 (a single occasion).

Statistical analysis. Multiple-comparison analysis of variance was performedusing Tukey’s family error approach on the growth curve data. Significance wasassessed at the 5 and 1% levels.

Antisera and GI tests. Bovine sera NK6 (Complement Fixation [CF] titer,1:640) and N28 (CF titer, 1:320) were gifts from Willie Amanfu, (NationalVeterinary Laboratory [NVL], Gaborone, Botswana) and came from unvacci-nated, naturally infected cattle (Botswana). Rabbit hyperimmune sera againstMmmSC strains were produced by two subcutaneous injections of inactivatedmycoplasma in adjuvant (Montanide ISA50), followed by one intravenous injec-tion of an aqueous suspension. Growth medium containing porcine serum was

used to culture the mycoplasma prior to immunization; for all subsequent ma-nipulations mycoplasmas were grown in medium containing horse serum tominimize cross-reactivities to serum components. Two rabbits were immunizedfor each mycoplasma strain, and sera were tested individually and pooled (resultsfrom all serum samples were highly similar for each strain tested). Rabbit hy-perimmune M. capricolum subsp. capripneumoniae antiserum was raised againsttype strain F38 using the same procedure as that described for MmmSC strains(this serum has been described in detail [19]). Bovine serogroup 7 antiserum(rabbit hyperimmune) was obtained from Aarhus, Denmark. For preabsorptionof antisera with capsular polysaccharide (CPS), 10 mg of purified CPS (22) wasadded to 0.1 ml of serum and the mixture was left at room temperature for 1 hand clarified by centrifugation. This procedure was repeated four times until theCPS antibody titer dropped to background levels (measured using a CPS en-zyme-linked immunosorbent assay). Growth inhibition (GI) tests were per-formed by spotting 20 ml of undiluted serum onto 5-mm-diameter filter paperdiscs (Mast Diagnostics, Merseyside, United Kingdom), placing the discs ontosuitable dilutions of mycoplasma cultures, and incubating at 37°C for 4 days priorto measurement of the zone of inhibition under a light microscope.

Western blot analysis. Whole mycoplasmas were pelleted from growth me-dium, washed twice in phosphate-buffered saline (PBS) (supplemented with 5%[wt/vol] glucose to prevent cell lysis), and resuspended in 5 volumes of sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer(0.1 M Tris-HCl [pH 6.8], 40% [vol/vol] glycerol, 4% [wt/vol] SDS, 0.25% bro-mophenol blue, 2% b-mercaptoethanol). Samples were boiled for 5 min andseparated by SDS-PAGE using a 12% homogeneous polyacrylamide gel followedby electrophoretic transfer to nitrocellulose membranes (Hybond C-Pure; Am-ersham, Little Chalfont, United Kingdom) using standard techniques (4). Rain-bow markers (Amersham) were run alongside the samples. Efficiency of transferwas estimated by staining the membranes with 0.1% Ponceau red (Sigma) in 1%acetic acid solution, followed by destaining in 1% acetic acid. The membrane wasthen rinsed twice in PBST (PBS plus 0.05% Tween 20) and blocked in 5%(wt/vol) dry skim milk in PBST for 1 h before primary antibody was added at adilution of 1:400. The membrane was subsequently incubated for 1 h at roomtemperature and rinsed three times in PBST, and then secondary antibody(rabbit anti-bovine horseradish peroxidase [HRP] conjugate [DAKO, Cam-bridge, United Kingdom] or donkey anti-rabbit HRP conjugate [SAPU, Lanark-shire, United Kingdom]) was diluted 1:400 into 5% (wt/vol) dry skim milk inPBST, and the membrane was incubated for a further hour at room temperature.Three 5-min washes in PBST were performed before incubation of the mem-brane in 0.1 mg of diaminobenzidine (Sigma)/ml in PBST containing 0.1 ml of30% H2O2 per 100 ml of substrate solution.

Insertion sequence analysis. Standard procedures were used for DNA manip-ulations and agarose gel electrophoresis (33). DNA extraction and insertionsequence analyses were performed as previously described (3, 7). Briefly, DNA

TABLE 1. M. mycoides subsp. mycoides SC strains used in this study

Strain Host Location Date isolated Source

Recent field isolatesN6 Cattle/lung Nokaneng, Botswana Oct. 1996 NVLa

M375 Cattle/lung Mohembo, Botswana Nov. 1995 NVLa

Tan1 Cattle Pawaga, Tanzania May 1996 ADRIb

Tan8 Cattle/pleural fluid Kagera, Tanzania March 1996 ADRIb

Vaccine strainsh

T1SR (5PRV317)d Cattle Tanzania 1952d BVIc

T144 (6PRV316) Cattle Tanzania 1952 BVIc

KH3J Cattle Sudan 1940 VLAg

European strainsC425 Goat/lung Portugal 1993 LNIVe

B820/124 Cattle/prepucial wash Portugal 1991 LNIVe

6479 Cattle/lung Italy 1992 LNIVe

Other strainsGladysdale Cattle Australia Pre-1964f VLAg

Afade Cattle Chad 1968 VLAg

V5 Cattle Australia 1936 VLAg

a Obtained from Willie Amanfu.b Obtained from Benedict Lema, Animal Diseases Institute (ADRI), Dar-es-Salaam, Tanzania.c Current vaccine stocks, obtained from the Botswana Vaccine Industry (BVI), Gaborone, Botswana.d Strain T1SR is derived from strain T144. The original isolation date of T144 is shown.e Obtained from Rosario Goncalves, Laboratorio Nacional de Investigacao Veterinaria (LNIV) Lisbon, Portugal.f Exact date of isolation unclear, although the first published record appears to be in 1964, where it is referred to as the “standard” challenge strain (14).g Obtained from Robin Nicholas, Veterinary Laboratories Agency (VLA), Addlestone, United Kingdom.h Batch numbers are in parentheses.

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was extracted using guanidinium thiocyanate, ammonium acetate, and phenol-chloroform-isoamyl alcohol. Aliquots of this DNA (200 ng) were then digestedusing either HindIII or EcoRI, and the resulting fragments were separated on a0.7% agarose gel. A digoxigenin-labeled 1-kb ladder (Gibco-BRL) was used as amolecular weight standard. Southern blotting was performed to transfer thedigested DNA onto nylon membranes (Amersham; Hybond-N). Membraneswere then incubated with either IS1296 (3) or IS1634 (37) digoxigenin-11–dUTP-labeled insertion sequence probes (supplied by Joachim Frey) using standardconditions, prior to visualization following incubation of the membrane in ni-troblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate) solution.

RESULTS

Morphological data. MmmSC cultures were diluted to giveapproximately equal densities of cells and plated onto Gour-lay’s agar. The field isolates fell into two distinct types (Fig. 1).N6, Tan1, and Tan8 all gave large homogenous colonies, whileM375 produced a mixture of both small and large colonies(approximately 5% of the total were of the large colony type).These larger colonies of M375 were still noticeably smallerthan the average colonies of the other field strains (Fig. 1) andwere observed at both high and low colony densities. Subcul-turing of individual small or large colonies of M375 on Gour-lay’s agar resulted in progeny displaying the same heterogene-

ity of colony size and appearance. This morphology appears tobe unique to M375 and has not been observed in this labora-tory for any other strains of MmmSC tested (we have examinedupwards of 20 strains originating from Europe, Africa, andAustralia).

Growth rates of MmmSC strains. Strain M375 grew notice-ably better on ME agar medium than on Gourlay’s agar me-dium. Colonies were larger and homogenous in nature, al-though still smaller than colonies of all other strains ofMmmSC tested. This effect was not seen with any of the otherstrains, in which there was no noticeable difference in colonysize between the two media. ME agar is a specialist culturemedium formulated for the isolation and growth of nutrition-ally demanding mycoplasmal species, and this presumably in-dicates a more fastidious nutritional requirement for M375than for other strains. The basis for this difference is notcurrently known but is unlikely to be the low passage level forM375 since this strain was found to be at least four levels ofpassage higher than strains Tan1 and Tan8 when tested andsince poor growth on Gourlay’s agar was still observed whenusing higher-passage stocks of M375 (data not shown).

To obtain a more quantitative estimate of the growth rate ofstrain M375, growth curves with GB were performed and thedoubling times of various strains were measured (Table 2).Strain M375 grew significantly more slowly than the otherstrains (P , 0.01 with the exception of KH3J, for which P ,0.05), with a mean doubling time of 346 min. For most otherstrains the doubling times were in the range of 168 to 229 min.The only exception was Gladysdale, with a mean doubling timeof 101 min, significantly faster than the other strains (P ,0.01). It is unclear if this rapid growth rate is due to adaptationto growth in vitro or whether this might indicate increasedvirulence for this strain (Gladysdale was used as a standardchallenge strain in Australia during the 1960s [14]). The addi-tion of 0.2% sodium pyruvate to the growth medium (shown tobe necessary for growth of many strains of M. capricolumsubsp. capripneumoniae (11), did not improve the growth rateof M375 (data not shown).

GI tests. GI activities of pooled pairs of antisera are shownin Table 3. Results are shown for rabbit hyperimmune seraraised against vaccine strain T1SR and field strains N6 andM375. Also shown are results observed using bovine sera NK6and N28 from field cases of CBPP (Botswana). Antisera weretested against the four field strains and two vaccine strains(T1SR and T144). An examination of the GI activity of rabbithyperimmune sera showed that test strains broadly appearedto fall into three categories with respect to their sensitivities toGI antisera: relatively insensitive (vaccine strains T1SR and

FIG. 1. Morphology of MmmSC recent field isolates grown on Gourlay’sagar. Cultures were diluted to give approximately equal colony densities andgrown for 3 days at 37°C prior to photography at equal magnifications. Botswanastrain M375, the “atypical” field isolate of the group, is shown in the lower rightpanel. Strain M375 morphology was noted at both high and low colony densities.

TABLE 2. Doubling times of various strains ofMmmSC in GB at 37°Ca

Strain Source Mean doubling time(min) 6 SD

M375 Africa, recent isolate 346 6 44KH3J Africa, vaccine strain 228 6 44B820/124 Europe 201 6 19N6 Africa, recent isolate 168 6 126479 Europe 189Gladysdale Australia, challenge strain 101 6 14

a M375, KH3J, B820, and N6, three growth curves; Gladysdale, five growthcurves; 6479, one growth curve. Gladysdale exhibited a significantly faster dou-bling time than KH3J, B820, N6, and M375 (P , 0.01). M375 was significantlyslower than B820, N6, and Gladysdale (P , 0.01) and KH3J (P , 0.05). Strain6479 was not included for statistical analysis.

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T144), medium sensitivity (field strains Tan1, Tan8, and N6),and highly sensitive (M375). This classification is most obviouswith N6 antiserum, although a similar pattern is also observedwith T1SR antiserum. M375 antiserum has very poor GI activ-ity, and in these tests only strain M375 itself exhibited sensi-tivity to this serum. In all instances strain M375 was the mostsensitive to the inhibitory effect of hyperimmune antiserum; asimilar pattern is also observed using bovine convalescent seraNK6 and N28, in which strain M375 is the only strain to showany growth inhibition. In a previously published report (22)M375 was shown to be the most sensitive to GI antisera out ofnine African and Australian strains. The increased sensitivityof strain M375 to GI antisera was not simply a function of thepoor growth of this strain on Gourlay’s agar compared to thatof the other strains, since an identical zone of growth inhibitionwas observed when the strain was grown on ME agar, in whichconsiderably better growth was seen (data not shown). Inter-estingly, the strain which is the most sensitive to growth inhi-bition (M375) is also the least effective at raising GI antiserum,while strain T1SR (relatively insensitive to GI) produces anti-serum with the best GI properties. Strains were not moresensitive to homologous antisera.

Rabbit hyperimmune serum raised against M375 also dis-plays some GI activity against M. capricolum subsp. capripneu-moniae. None of the other strains of MmmSC tested showedthis effect (Table 4). The GI activity of M375 antiserum wasqualitatively different from that observed using antiserumraised against M. capricolum subsp. capripneumoniae itself (orthat raised against bovine serogroup 7) in that a reduction incolony size rather than complete inhibition of growth was ob-

served. While cross protection using antisera raised againstthese two Mycoides cluster subspecies has been known forsome time (due to the highly similar or identical CPS sharedbetween M. capricolum subsp. capripneumoniae and bovineserogroup 7 [2, 32]), we believe that this is the first time thatantiserum raised against MmmSC has demonstrated GI activ-ity against M. capricolum subsp. capripneumoniae. These find-ings suggest a degree of relatedness between GI-sensitiveepitopes of M375 and M. capricolum subsp. capripneumoniaenot observed with other strains of MmmSC tested.

Immunoblot analysis. Strains of MmmSC were examinedfollowing SDS-PAGE and Western blotting for strain-specificpolymorphisms. Blots were probed both with bovine field an-tiserum (isolated from infected cattle from the recent outbreakin Botswana) and rabbit hyperimmune sera raised against theindividual strains.

(i) Bovine field serum. Nine strains of MmmSC (vaccinestrains T1SR and T144, recent field strains M375, N6, Tan1,and Tan8, and three older isolates, Gladysdale, Afade, and V5)were probed on a Western blot with NK6 bovine serum (Fig. 2,top). The serum identified a 110-kDa band present in T144,T1SR, Tan8, and M375 but either absent or present at a verymuch reduced intensity in the other strains. Interestingly,strain M375 displayed two unique polymorphisms: the absenceof a common 40-kDa band and the sole presence of a 70-kDaband. An identical banding pattern was observed with bovineconvalescent serum N28 when the strain was tested by Westernblotting (data not shown).

(ii) Rabbit hyperimmune sera. A mixture of the nine strainsof MmmSC described above was run as a single lane on aprotein gel, electroblotted, and probed with antisera raisedagainst the individual strains (Fig. 2, bottom). In agreementwith the immunoprofiles shown in Fig. 2, top, antisera raisedagainst M375 did not recognize a protein of 40 kDa in theantigen mixture, in contrast to antisera raised against the otherstrains. These data again agree with the premise that M375 isdistinct from the other three recent field strains (N6, Tan1, andTan8). Perhaps more surprisingly, these three field strains ap-pear to be immunologically more closely related to the otherstrains (some dating back to 1936) than to the fourth recentfield isolate, M375.

Insertion sequence analysis. Recently, two insertion se-quences, IS1296 (3) and IS1634 (37), have been identified andsuccessfully used to differentiate various mycoplasmal strains.Both insertion sequences were used to analyze the MmmSCstrains used in this study. IS1634 analysis (Fig. 3, top) ofHindIII-digested MmmSC DNA indicates that M375 bears aclose evolutionary relationship to the other Botswanan strain,N6, with both strains exhibiting identical banding patterns. Incontrast, Tanzanian strains Tan1 and Tan8 exhibit bandingpatterns clearly different from those of the Botswanan strains,although identical to each other. The two different bandingpatterns observed with these strains in the 3- to 4.5-kb regionwere also noted in a previous study using IS1634 (37). In thisearlier study, strains Fatick (Senegal) and 9050-52911 (IvoryCoast) gave the same pattern as we observed with Botswananstrains N6 and M375, while Afade and B17 (both from Chad),KH3J (Sudan), 94111 (Rwanda), and 95014, T144, and T1SR(all from Tanzania) gave the same pattern as the freshly iso-lated strains from Tanzania (Tan1 and Tan8). The distributionof these strains suggests that the current Botswanan and Tan-zanian outbreaks may have had different origins, in agreementwith published epidemiological findings (24). The strains sim-ilar to the Botswanan isolates were isolated from northwestAfrica, whereas those resembling the recent Tanzanian strainswere from northeast and central Africa. CBPP is known to

TABLE 3. Sensitivity of field and vaccine strains of MmmSC grownon Gourlay’s agar medium to GI by hyperimmune and

convalescent antiseraa

Host animal forserum production

Strain or serumantiserum

raised against

Zone of clearance (mm)in GI test againstb:

M375 Tan1 Tan8 N6 T1SR T144

Rabbit T1SR 4.5 3.5 3.0 3.0 2.5 2.3Rabbit N6 3.0 2.2 2.0 2.0 0.5 0.5Rabbit M375 1.0 0 0 0 0 0Bovine NK6 1.5 0 0 0 0 0

N28 1.0 0 0 0 0 0Cumulative total

GI zone 12.0 5.7 5.0 5.0 3.0 2.8

a Data were obtained from pooled antisera (two samples for each antiserum)except for bovine antisera, which were tested individually. The sensitivity of eachstrain to antisera can be estimated by the extent of the zone of inhibition seen.Rabbit antisera were hyperimmune and were raised against the strains shown.Bovine antisera were obtained from field cases of CBPP in Botswana.

b Strains M375, Tan1, Tan8, and N6 are field isolates; strains T1SR and T144are vaccine isolates. M375 is an atypical field isolate.

TABLE 4. Growth inhibiting activity of MmmSC rabbithyperimmune antisera against M. capricolum subsp.

capripneumoniae strain 19/2 grown on ME agar medium

Organism against which antiserum raised Zone of clearance (mm)

M. capricolum subsp. capripneumoniae ............5 (clear zone)Bovine serogroup 7.............................................2 (clear zone)MmmSC

T1SR (vaccine strain) .....................................0C425 (European strain) .................................0M375 (Botswana strain) .................................3 (reduced colony size)N6 (Botswana strain)......................................0



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have entered Botswana from Namibia to the west (24), inagreement with the insertion sequence data presented here.

Despite its close relationship to Botswanan strain N6 asshown by IS1634 analysis, M375 could be clearly differentiatedfrom N6 by probing HindIII-digested DNA with labeled

IS1296 (Fig. 3, bottom) due to the absence of an insertionsequence in M375. The absence of this band is unlikely to bedue to the loss of a restriction site, since this would result in theinsertion sequence still being present but at a higher molecularweight (which was not observed). This band was present inevery other strain of MmmSC tested (European, African, andAustralian).

FIG. 2. (Top) Immunoblot profiles for recent field isolates of MmmSC com-pared with vaccine strains and older isolates. Whole-cell antigens of the strainsshown were run on SDS-PAGE gels and electroblotted prior to incubation withserum NK6 from a naturally infected animal from Botswana. Polymorphisms at110, 70, and 40 kDa are shown, the last two unique to M375. (Bottom) Immu-noblot analysis using rabbit hyperimmune sera raised against the nine strains ofMmmSC shown in the top panel. In this case an antigen mixture of all ninestrains was run as a single lane on a gel and probed with sera raised against theindividual strains (using mixed duplicate serum samples). Arrow, unique poly-morphism present in antisera raised against M375 (nonrecognition of a proteinat 40 kDa).

FIG. 3. (Top) HindIII-digested genomic DNA of recent field isolates probedwith IS1634. Regions of variability are boxed. Similar patterns are observed withthe two Tanzanian isolates (Tan1 and Tan8) and the two Botswanan isolates (N6and M375). (Bottom) HindIII-digested genomic DNA of various MmmSCstrains probed with IS1296. The band of approximately 8.5 kb missing in M375is boxed. Note that the band has not simply shifted in molecular weight, as wouldbe expected if the absence were simply due to the loss or gain of a restrictionenzyme site.



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DISCUSSION

Strains of MmmSC isolated from recent outbreaks of CBPPin Africa have been compared to vaccine strains and olderisolates. Results suggest that Botswanan field isolate M375differs from all other strains of MmmSC tested by a variety ofcriteria. Compared with other strains of MmmSC, observeddifferences include altered colony morphology and poorgrowth in vitro, unique polymorphisms following immunoblot-ting, a high degree of sensitivity to growth-inhibiting antisera invitro, some level of GI activity for M375 antiserum against M.capricolum subsp. capripneumoniae, and the absence of a bandfollowing genomic hybridization with insertion sequenceIS1296. Other distinctive features of this strain include lowCPS production (22), a relative inability to cause septicemia inmice (20), and unique sugar utilization patterns (13). Some ofthese characteristics, such as poor growth in vitro (25, 35) andinability to cause septicemia in mice (17, 31), are also exhibitedby M. capricolum subsp. capripneumoniae, with which M375shares some serological similarity (some cross protection in GItests).

Strain M375 lacks an IS1296 element present in every otherstrain of MmmSC tested, which could indicate that M375 isevolutionarily more ancestral than the other strains studied.Alternatively, this missing band could indicate that a deletionevent has taken place in M375, removing both the insertionsequence element and portions of the surrounding chromo-somal DNA. The latter explanation is more likely given thephenotypic characteristics of M375. The absence of a 40-kDaband following immunoblotting, together with the poor growthobserved for this strain, would be consistent with a deletionevent removing some chromosomal DNA (and possibly somebiosynthetic or metabolic transport capability).

When grown on a standard culture medium for MmmSC,M375 gave very poor growth compared to other strains anddisplayed a unique morphology. This characteristic was far lessobvious when M375 was grown on a specialist mycoplasmaisolation medium (ME medium), suggesting that M375 hasunusually fastidious nutritional requirements. MmmSC is gen-erally regarded as being easy to culture (28), and the poorgrowth of M375 bears some resemblance to that of M. capri-colum subsp. capripneumoniae, the causal agent of contagiouscaprine pleuropneumonia (34). Interestingly, antiserum raisedagainst M375 displayed some GI activity against M. capricolumsubsp. capripneumoniae. This effect was not observed usingantiserum raised against any other strains of MmmSC. Al-though the inhibitory effect was small compared to that ob-served with homologous M. capricolum subsp. capripneu-moniae antiserum, these data would suggest that M375 may bemore closely related to M. capricolum subsp. capripneumoniaethan other strains of MmmSC examined.

Compared to other MmmSC isolates, M375 proved to beunusually sensitive to GI with MmmSC rabbit hyperimmuneantisera raised against a variety of different strains. Preabsorp-tion of these antisera with MmmSC CPS removed most of theGI effect, indicating that CPS must be a target for GI antibod-ies in vitro. Earlier work by us (22) showed that the GI activityof rabbit hyperimmune serum correlates with its CPS antibodytiter, while the sensitivity of a strain to GI antiserum inverselycorrelates with the amount of CPS produced by that strain (i.e.,the more CPS produced by a strain, the less sensitive it is toGI). M375 produces up to sixfold less CPS than other strains ofMmmSC when grown in culture (22), indicating a likely causefor the increased sensitivity of this strain to GI using rabbithyperimmune antisera. Interestingly, substrate utilization stud-ies (13) showed that strain M375 differed from other isolates of

MmmSC (European and African) by its ability to metabolizeglucosamine and mannose, two CPS components (22). Oxida-tion of these sugars, rather than their incorporation into thecapsule, may result in the lowered CPS production of strainM375.

The low CPS production of M375 may explain its apparentinability to cause a mycoplasmaemia following experimentalinfection of mice; in a study by us (20), only 30% of miceinfected with M375 exhibited a mycoplasmaemia, compared to80 to 100% of mice infected with other MmmSC strains. Sim-ilarly, the duration of the mycoplasmaemia in M375-infectedmice was only 24 h, compared to 3 to 5 days with other strains.Additional support for this hypothesis comes from a studyusing cattle that were experimentally infected with a low-viru-lence strain of MmmSC (KH3J) (14), which apparently did notcause a mycoplasmaemia unless additional CPS was added tothe inoculum at the time of infection. In another report (26),following experimental infection of cattle with variants of thesame strain of MmmSC producing low and high levels of CPS,variants producing low levels of CPS were present only in thelungs, while variants producing high levels of CPS resulted in amore general septicemia, with mycoplasma being found inmany tissues. This is the more usual situation with CBPP,where MmmSC is found in many tissues and bodily secretions(5). The implication is that a strain producing low levels ofCPS, such as M375, would be restricted to the lungs followinginfection. A similar situation is seen during contagious caprinepleuropneumoniae, which is believed to be lung specific (25,34). Thus both the clinical presentation and the likelihood ofisolation of MmmSC from an M375-infected animal may bealtered. This latter point could be particularly significant giventhe apparently poor growth of strain M375 on conventionalculture medium.

GI results using bovine field convalescent sera isolated fromBotswana (the geographical source of strain M375 itself)showed that this strain alone was sensitive to the inhibitoryeffects of these antisera. In contrast to results observed withrabbit hyperimmune sera, preabsorption of bovine convales-cent sera with CPS did not remove all GI activity, stronglysuggesting that non-CPS (presumably protein) antigens werethe target for GI. If so, the implication is that strain M375exhibits some unique protective epitopes not shared amongother strains of MmmSC.

Immunoblot analysis did reveal some unique polymorphismsfor M375 compared to other strains of MmmSC (contempo-rary field isolates, vaccine strains, and older type strains): theabsence of a common 40-kDa band and the sole presence of a70-kDa band. That these field sera recognized the unique 70-kDa band present in M375 is significant, since it suggests thatthe cattle from which these sera were isolated were infectedwith a strain similar or identical to M375. No other strainsexhibit these polymorphisms. Whether these polymorphismsaffect either virulence or immunity is unknown and perhapsmerits further investigation, as CPS is the only virulence factoridentified to date for MmmSC (18, 22).

What is likely to be of more immediate value, however, is todetermine the virulence and pathogenicity of these newer fieldisolates and the ability of vaccine strains to protect againstthem. Data to assess how widespread this new variant is wouldalso be welcome. Vaccination using T1SR was reportedly in-effective in controlling CBPP in Botswana (24) and field re-ports of a new clinical morphology for CBPP in this regionhave been made (R. S. Windsor, personal communication),both of which would be consistent with a new biotype. SinceM375 is difficult to culture and may be restricted to lung tissue



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only, the effect of a new biotype on current diagnosis of CBPPin Africa remains to be determined.

ACKNOWLEDGMENTS

We are grateful to Roger Windsor, David G. E. Smith, David Wind-sor, Hugh Reid, Roger Miles, Duncan Brown, Gareth Jones, and AnjaPersson for many useful discussions and suggestions. We are particu-larly indebted to Willie Amanfu and Benedict Lema for the supply ofstrains and sera. Statistical analysis was performed by Iain McKendrickof Biomathematics and Statistics Scotland. We thank Jenny Harrisonand Helen Williamson for skillful technical assistance.

This work was funded by the UK Department for InternationalDevelopment Animal Health Programme.

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