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tibody responses of swine following infection withycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and. flocculare

o Carlos Gomes Neto a,1, Erin L. Strait a,2, Matthew Raymond a,3,jandro Ramirez a, F. Chris Minion b,*

erinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011,

ed States

terinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, United States

1. Introduction

Mycoplasma infections continue to be of concern toswine producers world-wide. While Mycoplasma hyopneu-

moniae is the etiologic agent of enzootic pneumonia and amajor contributor to porcine respiratory disease complex(Opriessnig et al., 2011; Thacker and Minion, 2012), many

T I C L E I N F O

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pted 8 August 2014

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A B S T R A C T

Several mycoplasma species possessing a range of virulence have been described in swine.

The most commonly described are Mycoplasma hyopneumoniae, Mycoplasma hyorhinis,

Mycoplasma hyosynoviae, and Mycoplasma flocculare. They are ubiquitious in many pig

producing areas of the world, and except for M. hyopneumoniae, commercial antibody-

based assays are lacking for most of these. Antibody cross-reactivity among these four

mycoplasma species is not well characterized. Recently, the use of pen-based oral fluids for

herd surveillance is of increasing interest. Thus, this study sought to measure pig antibody

responses and the level of cross-reactivity in serum and pen-based oral fluids after

challenge with four species of swine mycoplasmas. Four groups of four mycoplasma-free

growing pigs were separately inoculated with the different mycoplasma species. Pen-

based oral fluids and serum samples were collected weekly until necropsy. Species-

specific Tween 20 ELISAs were used to measure antibody responses along with four other

commercial M. hyopneumoniae ELISAs. Animals from all groups seroconverted to the

challenge species of mycoplasma and no evidence of cross-contamination was observed. A

delayed antibody response was seen with all but M. hyorhinis-infected pigs. Cross-reactive

IgG responses were detected in M. hyopneumoniae- and M. flocculare-infected animals by

the M. hyorhinis Tween 20 ELISA, while sera from M. hyosynoviae and M. flocculare-infected

pigs were positive in one commercial assay. In pen-based oral fluids, specific anti-M.

hyopneumoniae IgA responses were detected earlier after infection than serum IgG

responses. In summary, while some antibody-based assays may have the potential for

false positives, evidence of this was observed in the current study.

� 2014 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +1 515 294 6347; fax: +1 515 294 8500.

E-mail address: fcminion@iastate.edu (F.C. Minion).

Currently at 352 Food Science Complex, University of Nebraska,

oln, NE 68583, United States.

Currently at Merck Animal Health, 35500 W. 91st St, DeSoto, KS,

18, United States.

Currently at Department of Pathology, University of Wisconsin,

ison, WI 53715, United States.

Contents lists available at ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

://dx.doi.org/10.1016/j.vetmic.2014.08.008

8-1135/� 2014 Elsevier B.V. All rights reserved.

J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171164

of the hurdles in the control of mycoplasma-associateddiseases of swine in general can be directly attributedto the ubiquitous nature of these microorganisms inaddition to the difficulty in determining the true status ofan exposed pig population (Sibila et al., 2009; Thacker andMinion, 2012). In addition, humoral immune responses toM. hyopneumoniae infection, as measured by currentlyavailable assays, are not reliable indicators of colonizationor disease severity (Andreasen et al., 2000; Fano et al., 2012).

Other mycoplasmas found in swine include M. hyorhi-

nis, Mycoplasma hyosynoviae and Mycoplasma flocculare. Inparticular, M. hyorhinis and M. hyosynoviae are of growinginterest among producers and veterinarians. Septicemiafollowed by serosal inflammation in nursery pigs andpolyarthritis in growing pigs are associated with M.

hyorhinis and M. hyosynoviae infections, respectively(Thacker and Minion, 2012). Data from submissions toveterinary diagnostic laboratories indicate a rise inrecognized cases associated with these organisms, in partdue to improved molecular diagnostic assays (Gomes Netoet al., 2012). Mycoplasma flocculare is also recognized to bepresent in pig populations, but knowledge regarding itsprevalence or disease potential is lacking (Thacker andMinion, 2012). The primary interest in M. flocculare is for itspotential to induce cross-reacting antibodies to M.

hyopneumoniae (Bereiter et al., 1990).As diagnostics for mycoplasmas progress, the demand

for rapid and inexpensive tests increases. Antibody-basedassays are frequently targeted for development. In additionto serum-based ELISAs, the use of pen-based oral fluidsamples for diagnostic use is growing in the swine industry(Ramirez et al., 2012). Thus, it is important to betterunderstand the potential for cross-reactivity among thesemycoplasmas and determine their ability to impact assayspecificity as has been previously described (Ameri et al.,2006; Freeman et al., 1984). There are few sets of defined-status serum samples from pigs infected with only a singlespecies of mycoplasma and no similar pen-base oral fluidsamples to investigate these potential cross-reactions.Therefore, the goals of this study were to generate pairedserum and oral fluid samples from swine infected with asingle-species of mycoplasma, estimate the extent ofcross-reactivity between these mycoplasmas using bothcommercial ELISAs and in-house Tween 20 detergent-baseELISAs, and evaluate the use of pen-based oral fluids forantibody detection.

2. Materials and methods

2.1. Experimental design

Caesarian-derived colostrum-deprived (CDCD) Land-raceand Large White cross-bred pigs were obtained from acontract facility when they were approximately 5 weeks oldfor all but the M. hyosynoviae challenge studies. For theseparate M. hyosynoviae study, the pigs were received atabout 12 weeks of age. All pigs were acclimated for 1 weekprior to being assigned to study groups and challenged. Allanimals were randomly assigned per treatment usingthe ear tag number initially assigned to all animals. Parentaland litter effects were not taken into consideration for

randomization. The pigs were housed under strict bio-containment conditions at the Iowa State University animalresearch facilities.

2.2. General animal husbandry

The study was performed at a Biosafety Level 2 facilitylocated at the College of Veterinary Medicine of Iowa StateUniversity. Animal chores were carried out on a daily basisby Laboratory Animal Resources personnel according tothe facility biosafety protocol. Each room has an anteroomwhere all individuals were required to wear disposablepersonal protective equipment including: Tyvek coveralls,gloves, plastic boot covers, hairnet, and an N95 respirator.Pigs were fed a commercial feed with no antibiotics. Waterwas freely available ad libitum. The animal experimentalprotocol was approved by the Iowa State UniversityInstitutional Animal Care and Use Committee.

2.3. Challenge inoculum

For the M. hyopneumoniae inoculum, a lung homoge-nate was used as described previously (Opriessnig et al.,2004). The lung inoculum (LI-42) is described inTable 1. For the M. hyorhinis inoculum, a 1 ml frozen stockof a triple cloned field isolate (Table 1) was passed into50 ml of BHI-TS (pH 7.4) containing 0.005% 2,3,5-triphe-nyl-2H-tetrazolium chloride, as a visual indicator ofgrowth. This was incubated stationary at 37 8C for 4 daysresulting in a deep orange color change due to tetrazoliumreduction. Purity was then checked by plating a sampleonto blood and chocolate agars, and onto solid BHI-TS agar(Ross and Karmon, 1970). All plating was performed intriplicate. Quantification of inocula was performed viatriplicate 10-fold serial dilutions using 2 ml volumes ofBHI-TS with tetrazolium resulting in CCU/ml. For the M.

hyosynoviae inoculum, a 1 ml aliquot each of two differentfield strain stocks (Table 1) were inoculated into 5 ml ofDifco PPLO Horse Serum (D-HS) medium. These wereincubated at 37 8C for 8 days resulting in dense cultureswith high turbidity. One milliliter of each 5 ml culture wasthen passed into 25 ml of D-HS. Purity was verified byspread plating samples onto blood and chocolate agar.Similarly, a sample was plated onto solid beef heartinfusion-turkey serum medium (BHI-TS, pH 7.4) (Ross andKarmon, 1970). All plating was performed in triplicate.Quantification of inocula was performed via 10-fold serialdilutions in D-HS broth containing arginine and phenol redand measured as color changing units (CCU)/ml. For the M.

flocculare inoculum (Table 1), a frozen stock of 1.5 ml waspassed into 50 ml of modified Friis medium containingphenol red (Friis, 1975). This was incubated at 37 8C for10 days resulting in a dark yellow color. Purity waschecked as described above. The inoculum was usedwithout further quantification by CCU (Table 1).

2.4. Assessment of mycoplasma status and challenge

For the M. hyopneumoniae (Strasser et al., 1992), M.

hyorhinis (Kobisch, 1983), and M. flocculare (Strasser et al.,1992) challenges, twelve 6-week-old female CDCD pigs

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J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171 165

re placed in three challenge groups, four pigs per group.pigs were found to be mycoplasma negative to all fourcoplasmas before challenge by PCR performed by thea State University Veterinary Diagnostic Laboratory onal swabs. Two ELISA-based assays, the Oxoid IDEIAcoplasma hyopneumoniae EIA kit (Oxoid Limited,ingstoke, UK), and the Tween 20 assay (Bereiterl., 1990) were used to test for the presence of serum

ibodies against M. hyopneumoniae prior to challenge.mals were found to be negative by all tests. Thecedures used for the challenge varied according tovious studies and are listed in Table 1. Infections wereformed by snaring the pigs and administering half theculum into each nostril (intra-nasal inoculum) or the

amount at once intra-tracheally. All animals in theseee challenge groups were necropsied at 118 days post-llenge.The M. hyosynoviae challenge was performed intrave-sly (IV) as previously described (Hagedorn-Olsen et al.,9). Four 14-week-old female CDCD pigs were placed inh of the two challenge groups (Table 1). Animals weretrained using a snare and 2 ml of 1010 CCU/ml M.

synoviae was administered IV using a butterfly catheteran ear vein. For the negative control group, 2 ml ofdium was administered IV using the same procedure.booster antibody responses, pigs were re-inoculatedh 5 ml of 1010 CCU/ml M. hyosynoviae (using the samein they were initially challenged with) administered

intra-nasally (IN) 15 days post challenge (DPC) by snaringthe pigs and administering half the inoculum into eachnostril using a 6 ml disposable syringe. At 39 DPC, a thirdbooster with 10 ml of 1010 CCU/ml M. hyosynoviae (usingthe same strain they were initially challenged with) wasadministered IN. Blood was collected periodically afterchallenge according to the schedule in Table 2. All animalsin this challenge group were necropsied at 66 DPC.

2.5. Sample collection

2.5.1. Blood, nasal swabs and tonsil scrapings

Samples were collected at time points described inTable 2. Blood samples were collected using a single-usecollection system (Vacuum tubes, BD, Franklin Lakes, NJ,USA). Samples were centrifuged at 2000 � g for 10 min,after which serum was aliquoted into 5 ml tubes. A singlenasal swab was used to sample both nares, and the swabwas placed in a 5 ml polystyrene round bottom snap-captubes containing 2 ml of PBS. Tonsil scrapings werecollected by snaring the pig and holding the mouth openwith an oral speculum and scraping the surface of thetonsil using a sterilized, blunt stainless steel spoon with anelongated handle (Wills et al., 1997). Tonsil scrapingsamples were then collected from the steel spoon using a15 cm sterile nylon flocked swab (Puritan1) and placed in5 ml tubes containing 3 ml of sterile PBS. All samples werestored at �20 8C until assayed by PCR.

le 1

oplasma strains and challenge methods.

thogen Straina Passage (p)/media used for growth Route of infectionb Inoculum vol. ml/infectious dose (CCU/ml)

. hyopneumoniae 232 Lung inoculum/Friis Intra-tracheal 5/NAd

. hyorhinis 3420 p10/BHI Intra-nasal 10/102

. hyosynoviae 3491

3496

P6/Difco + horse serum IV and INc 17e/1010

. flocculare 4843 P6/Friis Intra-tracheal 10/NAd

Mycoplasma hyosynoviae strains 3491 and 3496 were selected based on the previous history of each isolate and their ability to grow in the selected

ia. Two animals were challenged with each strain. Horse serum was used to diminish the risk of anaphylactic shock upon challenge.

Route of infection references: M. hyopneumoniae and M. flocculare (Strasser et al., 1992); M. hyorhinis (Kobisch, 1983); M. hyosynoviae (Hagedorn-Olsen

l., 1999).

IV and IN—intravenous and intranasal challenge.

NA, not applicable. M. hyopneumoniae lung inoculum was used without titration. For M. flocculare, no titration was done on the culture.

A total volume of 17 ml (media containing bacteria) was used for the challenge in three consecutive dates (2 ml IV on day 0, 5 ml IN on day 15, and 10 ml

n day 39 to maximize the antibody response. Animals were challenged throughout with the same strain they received on day 0.

le 2

ection schedule for swine mycoplasma challenges.

mple type Day post-challenge

ruma �1, 8, 15, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118c

rumb �3, 0, 1, 4, 7, 14, 15, 24, 39, 42, 49, 66

n-based oral fluidsc �1, 8, 12, 15, 27, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 114, 118c

sal swabsd (challenge group)

. hyopneumoniae, M. hyorhinis, M. flocculare 0, 15, 22, 30, 38, 46, 54, 62, 70, 76, 86, 94, 102, 110, 118

. hyosynoviae 10, 18, 36, 42

Serum samples from M. hyopneumoniae, M. hyorhinis, and M. flocculare challenged pigs were collected and tested individually.

Serum samples from M. hyosynoviae challenged pigs were collected and tested individually.

Pen-based oral fluids samples collected from the group of four animals present in the pen. All four animals were allowed contact with the rope. A volume

5–20 ml was collected at each time point. No samples were collected for M. hyosynoviae challenged pigs.

Nasal swabs were collected and tested by species-specific PCR.

J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171166

2.5.2. Pen-based oral fluids

Samples were collected from M. hyopneumoniae-,M. hyorhinis- and M. flocculare-infected animals as described(Kittawornrat et al., 2012) at time points listed in Table 2. A5/800 cotton rope was cut to length so that the end wasshoulder-high to the animals and hung in the center of thepen.Ropeswereleft inplacefor20to25 minoruntil15–20 mlof fluid could be collected. To recover the oral fluid specimen,the bottom (wet end) of each rope was inserted into a largeplastic boot, and the oral fluid was manually extracted bysqueezing the rope. The sample was decanted into a 50 mldisposable centrifuge tube for storage. At least 15 ml of oralfluid sample was collected from each pen at each time point(pre- and post-challenge). Powder-free nitrile gloves wereworn during the collection process and changed betweeneach pen. Samples were stored at �20 8C until assayed.

2.6. Necropsy

Animals from M. hyopneumoniae, M. hyorhinis, and M.

flocculare challenged groups were necropsied at 118 DPC.Lungs were evaluated macroscopically at necropsy andmultiple pieces (2 cm � 2 cm) of lung tissue were collectedfrom the middle left lobe of all animals at necropsy.Specimens from each animal were stored separately informalin (10%) for fixation until processing for histopath-ological and immunohistochemical evaluation by theVeterinary Diagnostic Laboratory at Iowa State University.The bronchial alveolar lavage (BAL) samples were collectedusing 25 ml of PBS to flush the lumen content of the leftmiddle lobe of lungs from all pigs. After collection, sampleswere kept at �20 8C until real-time PCR by the Iowa StateUniversity Veterinary Diagnostic Laboratory was per-formed to test samples for all four mycoplasmas. Standardculture procedures using blood and MacConkey agar plateswere carried out with BALs collected from all animals tocheck for the presence of other bacteria.

2.7. Enzyme-linked immunosorbent assays (ELISA)

The Oxoid IDEIA M. hyopneumoniae EIA kit (OxoidLimited), the IDEXX M. hyo. Ab Test (IDEXX Laboratories,Inc. Westbrook, ME), the Synbiotics SERELISA1 M. hyop AbMono Blocking ELISA (Zoetis, Inc., Kansas City, MO), andthe HIPRA CIVTEST SUIS1 MYCOPLASMA HYOPNEUMO-NIAE test (HIPRA, Girona, Spain) were used to test all seraper manufacturer’s instructions. Sera were also tested induplicate for the presence of antibodies against the othermycoplasmas using an adaptation of a Tween 20 assaypreviously described for M. hyopneumoniae (Bereiter et al.,1990). In brief, the antigens (Table 3) for the ELISAs wereextracted from whole-cell mycoplasma cultures using aTween 20 detergent reagent (Bereiter et al., 1990). Allmycoplasmas were grown in their respective requiredmedia (Table 3). ELISA plates were prepared individuallyfor each organism. Briefly, wells of 96-well plates (NuncImmulon 2 HB, Thermo Fisher Scientific Inc., Waltham, MA,USA) were coated with 100 ml of 10 mg/ml of each antigenin carbonate-bicarbonate buffer (pH 9.6). Thereafter, plateswere incubated overnight at room temperature and then

described (Bereiter et al., 1990). For testing of pen-basedoral fluids samples, 150 ml of oral fluid was mixed with150 ml of Tris buffered saline, and then 200 ml of this finalsolution was added to the plate (Kittawornrat et al., 2012).One positive (serum from animals of known status) andone negative control sera plus a well containing PBS wererun on each plate as quality controls. The negative controlsera were prebleed sera from the pigs prior to challenge.

Antibody isotype responses were determined usingperoxidase-conjugated goat anti-pig IgA (Bethyl Laborato-ries, Inc.), goat anti-pig IgM (m) (KPL, Inc.) and mouse anti-pig IgG (1:500 for pen-based oral fluids and 1:10,000 forserum samples) (ICN Biomedicals, Irvine, CA, USA). The KPLABTS peroxidase substrate system (Gaithersburg, MD,USA) was used to develop the color. A stop solution (PRV gIAntibody Test, IDEXX Laboratories) was dispensed intoeach well, plates were read at 405 nm (Model AL310, Bio-Tek Instruments, Inc., Winooski, VT, USA) and the resultsgiven as optical density (OD). For each plate, sample resultswere adjusted by subtracting the OD of the negativecontrol. ELISA values more than three standard deviationunits above background were considered positive.

2.8. PCR and biochemistry

The PCR assays for all four mycoplasmas wereperformed by the Iowa State University VeterinaryDiagnostic Laboratory. M. hyopneumoniae and M. flocculare

real-time PCR assays were performed as previouslydescribed (Stakenborg et al., 2006; Strait et al., 2008). M.

hyosynoviae (Lauerman, 1998) and M. hyorhinis (designedby ISU-VDL) assay were validated in house by cross-checking primers specificity using a panel of mycoplasmaland non-mycoplasmal species (Strait et al., 2008). Forsensitivity assessment, two plasmid constructs (pIDTS-MART-Invitrogen) were built using a 160 bp and 392 bpregion of 16S rDNA of M. hyorhinis and M. hyosynoviae,respectively. Reactions were run separately as M. hyorhinis

and M. hyosynoviae annealing temperature were validatedas 59 8C and 63 8C, respectively. Sensitivity was deter-mined to 10 genome copies per organism with anefficiency of either reaction above 92%.

Protein concentrations were determined by the Bio-RadProtein Assay (Bio-Rad Laboratories, Inc., Hercules, CA).

3. Results

3.1. Necropsy and culture

At 118 days post challenge, no lesions were found in

Table 3

Mycoplasmas antigens used for ELISA plate preparation.

Bacteria Strain Media

used for

growth

Antigen

concentration

(mg/ml/well)

M. hyopneumoniae 232 Friis 10

M. hyorhinis BTS-7 BHI 10

M. hyosynoviae 2861 Difco + Turkey Serum 10

M. flocculare 27399 Friis 10

the lungs of pigs infected with M. hyorhinis or M. flocculare.

stored at �80 8C until use. The ELISA protocol has been

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J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171 167

gs from pigs challenged with M. hyosynoviae were notluated at necropsy. Immunohistochemistry was notformed on lungs from pigs challenged with either M.

rhinis or M. flocculare because of the absence ofcroscopic lesions and PCR negative results in BAL fluidsta not shown). The histological results of the animalscted with M. hyopneumoniae identified microscopicons suggestive of M. hyopneumoniae infection in all pigsllenged with M. hyopneumoniae, which in combinationh positive PCR results in BAL from all pigs confirmedive infection in that challenge group (Table S1). Samplesected prior to challenge were negative for all fourcoplasmas, and throughout the duration of the study, noence of cross-contamination was identified. In addition,

other swine pathogens were identified by culture.

Serology

The dynamics of the antibody response in the M.

pneumoniae-challenged pigs as measured by the TweenELISAs developed slowly and were low in magnitude. 1), although most of the pigs did demonstrate aitive ELISA result on day 94 following the challenge.itive serum IgG responses did not appear until day 30 inmal no. 147. Positive reactions developed on day 54 inmals no. 127 and 199 and day 102 in animal no. 143. 1A). The IgM and IgA responses were borderlineitive throughout the study, with the caveat that theoff values for the IgM and IgA ELISAs have not beenidated and the cutoff value of the IgG ELISA was used.The species-specific Tween 20 serological responses in

other mycoplasma-challenged animals are shown in. 2. The Tween 20 ELISAs had the corresponding antigenhe challenge. The cutoff value shown is the same ash the M. hyopneumoniae Tween 20 IgG ELISA. The M.

rhinis challenged animals developed detectable anti-ies the quickest after challenge, at approximatelyDPC (Fig. 2A). All four pigs seroconverted by day

although the magnitude of the signals was low; the sera not induce high OD values in any of the animals. For M.

synoviae challenged animals, a positive Tween 20 re-nse occurred in two animals at day 35; the other twomals never developed detectable antibodies (Fig. 2B).

M. flocculare challenged animals, two animals ser-nverted late in the infection (Fig. 2C, days 70 and 86).

other two did not have detectable antibody responsesmuch of the study, although the responses were slowlyng at the time of necropsy.The serum responses to M. hyopneumoniae infectionasured by commercial ELISAs are shown in Table 4 andsupplemental Tables S2 and S3. Table 4 shows the

marized commercial ELISA results from the M.

pneumoniae challenged pigs. Without exception, allthe commercial assays were capable of identifyingcted animals over time. Few of the sera were stronglyitive; most of the responses were borderline positive.

OXOID and HIPRA ELISAs both showed borderlineults by day 15 following challenge. The IDEXX ELISA did

show positive results until day 36. Only one animals borderline positive (animal 199) throughout the 118-

Table S2 shows the results of the M. hyosynoviae-challenged pigs. The IDEXX ELISA had a positive responselate in the infection (pig no. 105). Table S3 shows theresults with the sera from M. flocculare-challenged pigs.Again, the IDEXX ELISA had a positive response late in theinfection with pig no. 156 corresponding to the resultswith the M. hyopneumoniae Tween 20 ELISA and in pig no.128 on day 70. None of the sera from M. hyorhinis-

challenged animals showed a response above backgroundwith the four commercial ELISAs (data not shown).

Cross-reactivity as demonstrated by Tween 20 ELISAs isshown in supplemental Figs. S1–S4. Fig. S1 shows thereactions of the sera from M. hyopneumoniae challengedanimals with the other three mycoplasmas. Cross-reactiv-

Fig. 1. Anti-M. hyopneumoniae IgG, IgM and IgA responses in serum of pigs

experimentally challenged with M. hyopneumoniae. (A) IgG values; (B)

IgM values; (C) IgA values. The M. hyopneumoniae Tween 20 antigen

(strain 232) was used in this assay. Mouse anti-pig IgG and goat anti-

swine IgM and IgA conjugates were used. A cut-off OD value of 0.25 has

been previously validated for serum IgG and was used also for IgM and IgA

values. Thus, OD values above 0.25 are considered positive for all antibody

isotypes. Data represents the mean of duplicate wells for each animal.

was demonstrated with the M. hyorhinis antigen. All

study by the Synbiotics ELISA. ity

J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171168

four animals were close to seroconverting throughout thestudy and two animals did have positive cross-reactive ODvalues at the end of the experiment. There were no cross-reactive OD values to the M. hyosynoviae and M. flocculare

antigens.Supplemental Fig. S2 shows the response of sera from

M. hyorhinis challenged animals and antigens from theother three mycoplasmas. The only potential cross-reactivity was late in the infection with the M. flocculare

antigen. Fig. S3 shows cross-reactivity with the sera fromM. hyosynoviae challenged animals. Although no positivereactions were observed, late in the infection, two animalsapproached a positive response with the M. flocculare

antigen. Fig. S4 shows cross-reactivity with sera from M.

flocculare challenged animals. Cross-reactive responses areshown with the M. hyorhinis antigen. Borderline responseswere measured in one animal by day 22, a second by day 46

(animal no. 156). That animal did seroconvert andremained seropositive throughout the study.

3.3. Pen-based oral fluids

Antibody responses in pen-based oral fluids weredetected after M. hyopneumoniae infection (Fig. 3). Theearliest response was with IgA by day 20. The IgG responsedid not become positive in the oral fluids until day 66. Oralfluids from M. hyorhinis and M. flocculare-infected animalsshowed no positive responses throughout the course ofinfection by either the M. hyopneumoniae Tween 20 ELISAor the species-specific ELISAs (data not shown).

3.4. PCR analyses

Analysis of nasal swabs (M. flocculare and M. hyorhinis-challenged pigs) and tonsil scrapings (M. hyosynoviae-challenged pigs) was performed to confirm colonization.Nasal swabs became positive in four of four pigs in the M.

flocculare and M. hyorhinis challenged pigs at day 18, theonly day analyzed (data not shown). Only one of four pigsin the M. hyosynoviae challenged group had positive nasalswabs at day 18, however. When tonsil scrapings wereexamined, all four of the pigs were positive for M.

hyosynoviae by as early as day 7 (data not shown). Theseresults indicated that all pigs in the challenged groupswere colonized by their respective mycoplasma duringchallenge and remained positive for at least a week.

4. Discussion

Though advances in veterinary diagnostics have beenmade through the years, there is still a large dependence onELISA-based serological assays for diagnosis of M. hyop-

neumoniae infections in swine (Thacker and Minion, 2012).This includes the IDEXX M.hyop Ab Test, the Oxoid IDEIAM. hyopneumoniae EIA kit, the HIRPA CIVTEST SUIS1

MYCOPLASMA HYOPNEUMONIAE test and the SynbioticsSERELISA1 M.hyop Ab Mono Blocking ELISA and the Tween20 assay (Bereiter et al., 1990). These assays are rapid, buttheir results can be difficult to interpret, especially in cases

Fig. 2. IgG responses in serum of pigs experimentally challenged with M.

hyorhinis (A), M. hyosynoviae (B), and M. flocculare (C). The Tween

20 antigen corresponding to each strain listed in Table 3 was used in this

assay. Mouse anti-pig IgG conjugate was used. Data represents the mean

of duplicate wells for each animal.

Fig. 3. Anti-M. hyopneumoniae IgG, IgA, and IgM responses in pen-based

oral fluids of 4 pigs experimentally challenged with M. hyopneumoniae.

The M. hyopneumoniae ELISA antigen and secondary antibodies were as in

Fig. 1. Data represents the mean of duplicate wells for each animal.

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J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171 169

h routine use of vaccines, the presence of maternally-ived immunity, or when questions of cross-reactivitye.The most definitive method for diagnosis, culture, iscult and expensive, and more importantly, it requires

use of lung specimens or BAL fluids. Most diagnosticoratories do not offer this service due to the fastidiouswth of mycoplasmas, the time involved (weeks) and theential for overgrowth by other bacteria. Although thevenience aspect of serological assays is clear, M.

pneumoniae ELISAs suffer from a low sensitivityulting in low negative predictive values (Bereiteral., 1990; Erlandson et al., 2005; Okada et al., 2005)ble 4). In addition, M. flocculare antibodies have beenorted to cross-react with M. hyopneumoniae (Bereiteral., 1990; Freeman et al., 1984). One of these cross-ctions is characterized by the monoclonal antibody5, which binds to the ciliary adhesin of M. hyopneu-

niae (Zhang et al., 1995) and also cross reacts with M.

culare (Strait and Minion, unpublished). Antibodyponses are known to be poorly predictive of diseaseus, and concurrent infection with PRRSV (Thacker et al.,9), SIV (Thacker et al., 2001), or PCV2 (Opriessnig et al.,4) appear to increase M. hyopneumoniae antibody

els. Thus, these ELISA assays are best used to predict M.

pneumoniae infection on a herd basis rather than anividual basis.In the case of other swine mycoplasmas such as M.

rhinis, M. hyosynoviae or M. flocculare the absence of aidated assay is an obstacle. Adapting the methodcribed for the M. hyopneumoniae Tween 20 ELISAvided a useful measure of antibody responses, thoughse assays have not been validated for use in a diagnosticoratory setting.The impact of cross-reactivity of the various swinecoplasmas on the various commercial M hyopneumo-

e ELISA assays is not well characterized. Our resultsicate that cross-reactivity can occur with the IDEXXSA, which showed positive responses in a few animals

both M. hyosynoviae and M. flocculare-challengedups (Tables S2 and S3). The M. hyopneumoniae Tweenassay did not show cross-reactivity against the sera

the other three challenge groups (Figs. S2–S4). Thisy be due to its lower sensitivity of the latter.It should be noted that mycoplasma disease could be

onstrated in the M. hyopneumoniae and M. hyosynoviae

llenge groups, but not the M. hyorhinis and M. flocculare

llenged groups. Our PCR results clearly show that thecoplasmas were present in the challenged pigs afterays or more, and the serological responses suggest thatction had occurred as well. Failure to demonstrateons at necropsy in the M. hyorhinis and M. flocculare

llenged pigs is likely due to the clearance of thesections from the lungs by the time of necropsy 118 dayst challenge or the lack of sensitivity of our assays toect them. It should be noted that there are no wellblished infection models for M. hyorhinis, M. hyosyno-

and M. flocculare. Ross et al. (1971) gave M.

synoviae intravenously and were able to induceritic lesions. Magnusson et al. (1998) reported diseaser intraperitoneal injection with M. hyorhinis. Freeman

et al. reported a study infecting CDCD pigs intranasallywith M. flocculare (Armstrong et al., 1987), but they hadsimilar results to ours, inability to re-isolate the organismand low serological responses.

Of the four mycoplasmas, the most difficult toseroconversion with was M. hyosynoviae (Nielsen et al.,2005). These animals were re-challenged overtime with asecond field strain (Table 1) to induce better antibodyresponses. It should be noted that these animals were on adifferent schedule (necropsy was on day 66) than the otherthree mycoplasmas (necropsy on day 118). This was due tothe fact that this challenge was the first to be performed,and the decision to extend the necropsy date was madeafter this initial experiment. Perhaps if we extended thetime frame of the M. hyosynoviae challenge experiment, wewould have had seroconversion in all four animals .

This study was designed to follow the dynamics ofantibody responses to different swine mycoplasmas anddetermine the extent of cross-reactivity between theseresponses as measured by ELISA assay. One criticalcomponent of this research was the mycoplasma andother pathogen status of the pigs. We carefully followedthe mycoplasma status throughout the experiment and,without exception, could show that these animalsremained mycoplasma-free through the experiment ex-cept for the infecting species. As the number of animalsavailable for such studies is an obstacle due to the requiredstatus, randomization could not be done by litter orparental history since a very limited number of littermateswere available. Therefore, our results are a reflection of ourexperimental setting requiring further investigation usingclinical and experimental cohorts with a larger number ofanimals for confirmation. The bacterial status of the pigswas assessed upon necropsy by plating BAL fluids as wellas testing nasal swabs and other tissue samples. Thus, wehad confidence that any cross-reactivity shown with theserum was due to true antigenic similarities among themycoplasmas and was not due to a low-level mycoplasmainfection of another species. However, variation and lowlevel of antibody responses in some cases may beattributed to the potentially immature immune systemof CDCD animals.

When tested by commercial M. hyopneumoniae ELISAs,the sera developed during the course of this study to othermycoplasma species showed some evidence of cross-reactivity (Tables S2 and S3). Perhaps it would have beenmore extensive if a more robust infection had beenestablished as is seen under natural conditions. Since thegoal of this study was to develop the mycoplasma-specificswine sera for analysis, we were limited to CDCD pigs fromfarms that had been monitored extensively for mycoplas-ma infections to ensure that our animals would bemycoplasma free. It would be useful to repeat thisexperiment with a wider range of field isolates, as well.

Considering the ease with which the pen-based oralfluids can be collected on the farm, this sample has promiseas an option for assessing the presence of M. hyopneumo-

niae on a herd basis by ELISA. In support of that,M. hyorhinis and M. hyosynoviae have been demonstratedto be present in this sample type in naturally exposedpig populations Gomes Neto, unpublished). Additionally,

J.C.G. Neto et al. / Veterinary Microbiology 174 (2014) 163–171170

pen-based oral fluids have previously been successfullyutilized to identify other swine bacterial and viralpathogens (Bender et al., 2010; Costa et al., 2012; Prickett

and Zimmerman, 2010; Ramirez et al., 2012). However,further evaluation and validation of these diagnosticassays is required before use can begin.

5. Conclusion

In conclusion, we have produced the first complete setof sera from mycoplasma-free swine infected with the fourdifferent swine mycoplasma species. When analyzingthese sera on a pig by pig basis with five differentcommercial ELISA assays, we observed low antibodyresponses with M. hyopneumoniae challenged animals.The least sensitive assay was that from Synbiotics whereonly one animal seroconverted according to the assayinstructions potentially resulting in a large percentage offalse negative calls. The delayed antibody detection thoughmay be of concern, but yet it is an indication thatsurveillance of farms may be more productive and easierto be interpreted when selecting older animals in growingand finishing units. Depending on the assay used, lowpositive reactions could also reflect M. hyosynoviae or M.

flocculare infection as well. The IgA response, whichparallels pig colonization status in pen-based oral fluidsamples should be further explored and could provide asensitive, cost-effective surveillance tool after furtheroptimization. Collectively, these findings demonstratedthat sera cross-reactivity among all major swine myco-plasmas can occur; however, subsequent studies using alarger number of animals in experimental and clinicalsettings will be needed to further validate the analyticalimpact of such assays. Additionally, a broader panel ofstrains will have to be used for characterization ofantigenicity and immunogenicity if more sensitive andspecific immune assays are to be developed. Nonetheless,our experimental approach becomes an important modelfor such validations since the status of exposure before andduring the study is crucial for its success.

Acknowledgements

Funding, wholly or in part, was provided by Inc.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2014.08.008.

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Table 4

M. hyopneumoniae challenged pigs ELISA results*.

Pig number DPC* OXOID HIPRA IDEXX Synbiotics

127 �1 0 0 0 0

143 �1 0 0 0 0

147 �1 0 0 0 0

199 �1 0 0 0 0

127 8 0 0 0 0

143 8 0 0 0 0

147 8 0 0 0 0

199 8 0 0 0 0

127 15 2 0 0 0

143 15 1 1 0 0

147 15 1 0 0 0

199 15 1 0 0 0

127 22 1 1 0 0

143 22 1 1 0 0

147 22 1 2 0 0

199 22 1 1 0 0

127 30 1 1 0 0

143 30 1 1 0 0

147 30 1 1 0 0

199 30 1 1 1 0

127 38 1 1 1 0

143 38 1 1 2 0

147 38 1 1 1 0

199 38 1 1 1 1

127 46 1 1 1 0

143 46 1 1 1 0

147 46 1 1 1 0

199 46 1 1 1 1

127 54 1 1 1 0

143 54 1 1 1 0

147 54 1 1 1 0

199 54 1 1 1 1

127 62 1 1 1 0

143 62 1 1 1 0

147 62 1 1 1 0

199 62 1 1 1 1

127 70 1 1 1 0

143 70 1 1 1 0

147 70 1 1 1 0

199 70 1 1 1 1

127 78 1 1 1 0

143 78 1 1 1 0

147 78 1 1 1 0

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127 86 1 1 1 0

143 86 1 1 1 0

147 86 1 1 1 0

199 86 1 1 1 1

127 94 1 1 1 0

143 94 1 1 1 0

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127 102 1 1 1 0

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147 102 1 1 1 0

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127 110 1 1 1 0

143 110 1 1 1 0

147 110 1 1 1 0

199 110 1 1 1 1

127 118 1 1 1 0

143 118 1 1 1 0

147 118 1 1 1 1

199 118 1 1 1 1

* 0 = positive, 1 = positive, 2 = suspect; DPC = days post challenge.

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