Vertical transmission of Mycoplasma haemolamae in alpacas (Vicugna pacos)

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Small Ruminant Research 106 (2012) 181– 188

Contents lists available at SciVerse ScienceDirect

Small Ruminant Research

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ertical transmission of Mycoplasma haemolamae in alpacasVicugna pacos)

ebecca L. Pentecosta, Antoinette E. Marsha, Andrew J. Niehausa, Jackeline Daleccioa,oshua B. Danielsa, Paivi J. Rajala-Schultzb, Jeffrey Lakritza,∗

Department of Veterinary Clinical Sciences, The Ohio State University, College of Veterinary Medicine, 601 Vernon L Tharp Street, Columbus, OH 43210,nited StatesDepartment of Veterinary Preventive Medicine, The Ohio State University, College of Veterinary Medicine, 1920 Coffey Road, Columbus, OH 43210,nited States

r t i c l e i n f o

rticle history:eceived 16 October 2011eceived in revised form 23 January 2012ccepted 28 February 2012vailable online 27 March 2012

eywords:ycoplasma haemolamae

CRamria

gGFAT

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The purpose of this study was to determine the frequency of vertical transmission ofMycoplasma haemolamae from dam to cria, whether colostral transmission of M. haemola-mae occurs and provide preliminary data on colostral M. haemolamae specific antibody frompregnant alpacas on a farm with known prevalence of infection. M. haemolamae specific PCRwas performed on blood and colostrum from pregnant alpacas and their cria (n = 52 pairs).Indirect fluorescent antibody testing was performed on a subset (n = 43) of the colostrumsamples. Total immunoglobulin concentrations of colostrum and cria sera and M. haemola-mae specific IgG (prior to and after ingesting colostrum) were determined by turbidometricimmunoassay and indirect fluorescence antibody testing respectively. Sixteen of 52 dams(30.7%) pre-partum and one of 52 cria post-partum (1.9%; prior to ingesting colostrum)were PCR positive for M. haemolamae, while 36/52 dams (69%) and 51/52 cria (98%) testednegative for M. haemolamae by PCR. All 43 colostrum samples and 52 of 52 post-colostrumcria blood samples (100%) were negative by PCR. The dam giving birth to the M. haemolamaePCR positive cria was PCR negative. Statistically, it was no more likely for a PCR positivedam to give birth to a M. haemolamae, PCR positive cria (prior to colostrum ingestion) thana PCR negative dam (p = 0.3077). M. haemolamae specific IgG was present in 22 of 43 (51%)of colostrum samples at a 1:10 dilution and 14 of 22 (64%) at a 1:100 dilution. There was norelationship between the PCR status of the dam and whether or not M. haemolamae specificantibodies were present in colostrum. Among the animals tested, in utero transmission ofM. haemolamae was rare (1/52 pre-colostral alpaca cria), and all colostrum samples werenegative for M. haemolamae by PCR. These data indicate that colostrum from positive damsis unlikely to harbor this parasite and therefore does not serve as a source of infectionto newborn cria. Colostrum derived from both PCR positive and negative dams containedM. haemolamae specific antibodies. Our findings suggest that M. haemolamae specific anti-

d dete ble titers may provide different estimates than those available

∗ Corresponding author. Tel.: +1 614 292 6661x48696; fax: +1 614 292 3530.E-mail address: Jeffrey.Lakritz@cvm.osu.edu (J. Lakritz).

921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.smallrumres.2012.02.021

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© 2012 Elsevier B.V. All rights reserved.

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1. Introduction

Mycoplasma haemolamae is a hemotropic parasite of lla-mas, alpacas, and guanacos. Currently, the biological ormechanical vector(s) responsible for transmission of thisorganism are unknown; however, it has been hypothesizedthat biting insects transmit this agent in some herds. Exper-imentally, transfusion of M. haemolamae infected bloodwas found to transmit this organism (Tornquist et al., 2009).In that study, animals did not test positive by PCR untilat least 4 days post-transfusion, indicating that the timingof sample acquisition may be important for detecting andstudying neonatal infection (Tornquist et al., 2009, 2011).

Three reports, each involving very low numbers ofanimal subjects (n ≤ 5) have suggested that in utero trans-mission of M. haemolamae is possible, while acquisition ofthis parasite via consumption of colostrum by the new-born cria was not observed (Almy et al., 2006; Fisher andZinkl, 1996; Tornquist et al., 2011). A recent prospectivestudy evaluated in utero transmission of M. haemolamaefrom 5 PCR-positive pregnant dams to their cria. This studydetected 2 PCR-positive crias by evaluating blood for M.haemolamae DNA prior to colostrum ingestion (Tornquistet al., 2011). One colostrum sample consumed by one criawas PCR negative for M. haemolamae. In each of the 2 M.haemolamae PCR-positive cria, post-colostrum blood sam-ples remained PCR positive for several months (Tornquistet al., 2011).

Since the epitheliochorial placentation of camelidslimits the exchange of most macromolecules (e.g.immunoglobulins), between the dam and the fetus,colostral acquisition of maternal antibodies is importantto the health of the newborn (Fowler and Olander, 1990;Steven et al., 1980; Bjorkman, 1973; Wernery, 2001;Weaver et al., 2000). While concentrations of IgG innormal camelid colostrum have been described (rangesbetween 176 and 360 mg/ml), little information regardingM. haemolamae specific antibody is available (Wernery,2001; Bravo et al., 1997). One study described serologicresponses of llamas infected with M. haemolamae; how-ever, PCR based testing methods were not available atthat time (McLaughlin et al., 1990). That seminal studyestimated a seroprevalence of 12% among 1753 animalsfrom 4 different states; however, the presence of M.haemolamae specific antibodies in pregnant camelids,colostrum and cria was not evaluated (McLaughlin et al.,1990).

M. haemolamae, was originally described in 1990 asan Eperythrozoon-like organism, and is currently classi-fied among the hemoplasmas based upon morphologic andmolecular similarities (McLaughlin et al., 1990; Messicket al., 2002). Genetically, M. haemolamae is highly homolo-gous to Mycoplasma suis and Mycoplasma wenyonii, whichaffect swine and cattle, respectively (Reagan et al., 1990;McLaughlin et al., 1990; Messick et al., 2002). The sequenceof the M. haemolamae 16S ribosomal RNA (rRNA) geneis known and is reported to be a useful target for the

esearch 106 (2012) 181– 188

detect M. haemolamae from camelids from North and SouthAmerica, Europe, and Australia, suggesting that 16S rRNAsequence is conserved among isolates from different geo-graphic regions (Tornquist et al., 2009, 2010; Kaufmannet al., 2010).

Polymerase chain reaction assays are widely used todiagnose infections of numerous hemotrophic Mycoplasmaspecies (Tornquist et al., 2009; Meli et al., 2010; Sykeset al., 2007; Hoelzle et al., 2003). The limit of detection ofPCR based assays has been reported to range from 1 to 28copies per reaction (Meli et al., 2010; Tornquist et al., 2009).Therefore, PCR may be useful as a tool for clinical diagnosisas well as prevalence of infection. Prevalence rates deter-mined using PCR based assays vary between study andgeographic regions with a range of 9.3–19.3% for camelidsin South America, and a prevalence of 18.6% with nearly40% of tested herds having at least one positive animal inSwitzerland (Tornquist et al., 2010; Kaufmann et al., 2010).

Clinically, individual animals infected with M. haemo-lamae may be variably anemic and have additional clinicalsigns: lethargy, exercise intolerance, weight loss, and infre-quently hypoglycemia (Reagan et al., 1990; Fisher andZinkl, 1996; Lascola et al., 2009; Barrington et al., 1997).Clinical hemoplasma infections are reported in young orimmunocompromised adult camelids (Reagan et al., 1990;McLaughlin et al., 1990; Meli et al., 2010; Almy et al.,2006; Tornquist et al., 2010; Fisher and Zinkl, 1996; Lascolaet al., 2009; Barrington et al., 1997). It is reported that themajority of infected animals never develop clinical disease(Tornquist et al., 2009).

The purpose of this study was to determine whether inutero or colostral transmission of M. haemolamae is a signif-icant route of infection for newborn cria. We hypothesizedthat in utero or colostral transmission of M. haemolamaeleads to detectable infection in newborn cria as defined bya standard PCR assay. A second objective was to evaluatethe presence of M. haemolamae specific antibodies in damswith known PCR status for this organism. We hypothesizedthat PCR positive dams produce higher concentrations ofM. haemolamae specific antibody in colostrum when com-pared to PCR negative dams.

2. Materials and methods

2.1. Dam–cria pairs

All procedures involving animals and sample collection wereapproved by the Institutional Animal Care and Use Committee at TheOhio State University, the Clinical Trials Committee of the OSU VeterinaryMedical Center and were performed after obtaining the owner’s consent.Samples were collected from 67 dams and their cria housed on an alpacabreeding farm in southwestern Ohio between October 2009 and August2010. During the years from 2005 to 2008, no whole blood transfusionswere performed on any animals from this farm, while 5 plasma transfu-sions were performed. Two of these plasma transfusions were performedon cria admitted to our hospital and the remaining 3 were performed bythe referring veterinarian on the farm. All plasma transfusions utilizedcommercial Llama plasma.

Each sample set contained a blood sample (EDTA anti-coagulated)from the dam at the time of parturition (A), a blood sample from the

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ll alpacas were closely monitored during parturition to ensure thathe cria were not able to nurse prior to collection of a pre-colostrallood samples. Samples were placed in EDTA blood collection tubes andefrigerated at 4 ◦C prior to analysis. Data from 12 of the 67 dam–criaairs were not included as either pre-colostral (n = 8) or post-colostralria blood samples (n = 4) were not available. In addition, 3 sets wereithdrawn due to failure to extract sufficient DNA or the presence of PCR

nhibitors in the corresponding colostrum sample. Thus, PCR analysisas performed on blood from the remaining 52 complete sample sets.

erum immunoglobulin (IgG) data both prior to ingesting colostrumnd 72 h post-colostrum was available for all 52 cria included in thistudy. Sufficient volumes of colostrum for determination of colostral IgG,. haemolamae and indirect fluorescent antibody testing (IFAT) were

vailable for 43 of the 52 dam and cria pairs.

.2. DNA extraction and PCR

DNA was prepared from 50 �l whole EDTA-anticoagulated bloodsing a commercial preparation kit (DNeasy Blood and Tissue kit; Quia-en Corp, CA, USA) with minor modifications to the extraction protocol.amples were placed into micro-centrifugation tubes containing 20 �l ofroteinase K, 220 �l of PBS and 200 �l of AL buffer extraction buffer. Theame kit was used to prepare DNA from 50 �l colostrum samples, how-ver, due to sample viscosity of 3 colostrum samples, 40 �l of proteinase

was used to facilitate protein digestion. The sample-buffer proteinase Kolutions from blood or colostrum were pulse-mixed for 15 s and heatedo 56 ◦C with constant rotation for 20 min. The remaining steps were com-leted without modification.

Three different primer sets were used in this study (Table 1). To con-rm that each sample contained amplifiable template DNA for PCR, primeret I (Table 1) was used as an internal control to amplify an alpaca-specificNA sequence. We anticipated some genomic alpaca DNA carry-over

leukocytes, epithelial cells) from blood or colostrum to serve as this con-rol template. This primer pair was originally designed to target a smallNA fragment of a conserved alpaca gene evaluated as part of a different

tudy.Primer set II (Table 1) amplified a 415-bp fragment of M. haemolamae

NA, targeting the 16S ribosomal RNA (rRNA) gene. For each reaction,.25 �M of each primer, and 1 �l of DNA template were added to 23 �l of

commercially available PCR mastermix (PCR Supermix, Invitrogen, CA,SA). Negative controls consisted of all reagents without a DNA template,nd positive controls consisted of all reagents with our previously con-rmed, PCR positive DNA sample extracted using the same commercialit. Cycling conditions for primer set I started with initial denaturation at4 ◦C for 2 min, amplification by 30 cycles of 94 ◦C for 30 s, 52 ◦C for 45 s and2 ◦C for 2 min. The final cycle consisted of 94 ◦C for 30 s and 52 ◦C for 30 sith a final elongation at 72 ◦C for 7 min. Cycling conditions for primer set

I were the same as for primer set I. The PCR products were evaluated byel electrophoresis on 2% (w/v) agarose gel (NuSieve, 3-1 Agarose, Lonza-ockland Inc., ME, USA) in Tris–Borate–EDTA (TBE) stained with ethidiumromide and viewed by ultraviolet transillumination. Approximate ampli-on sizes were estimated by running a 100 base pair DNA ladder on eachel.

To evaluate primer set I specificity, DNA was extracted from bovine,vine, equine, caprine, Dromedary camel, and llama ETDA anti-coagulatedlood. To evaluate primer set II specificity to M. haemolamae, reac-ions with template DNA prepared from infected blood or culturesontaining Mycoplasma haemofelis (Mhf), Mycoplasma haemominutum

able 1rimer sets, amplicon sizes and sequences used in this study.

Primer set Size (bp)

Set I SensegDNA-219 bpa

Set I Anti-sense

Set II SenseMh-415 bpb

Set II Anti-sense

Set III SenseMh-696 bp

Set III Anti-sense

a gDNA-219 bp indicates amplification of genomic DNA of host cells as amplicob Mh indicates Mycoplasma haemolamae directed primers for the 16S, small sub

esearch 106 (2012) 181– 188 183

(Mhm), Mycoplasma gallisepticum (Mg), Mycoplasma bovis (Mb), andMycoplasma synoviae (Ms), were performed. DNA was provided by Dr. JaneE. Sykes, University of California Davis, CA, USA (Mhf and Mhm); and Dr.Ziv Raviv, Dr. Amy Wetzel, The Ohio State University, Columbus, OH, USA(Mg, Mb, and Ms).

Sensitivity of the Mh-specific primer set (set II) was evaluated using alimiting dilution series of a known positive blood sample, thus the mea-surement of sensitivity was relative to a standard control sample (versusan absolute copy number). To evaluate the sensitivity of primer set II, serialdilutions from 1:10 to 1:100,000 of blood from the clinically affected cria,and the pre-colostral PCR-positive (in utero infection) cria were used, andDNA samples from these isolates were reacted with primer set II. Likewise,to evaluate the sensitivity of the PCR assay in colostrum samples, PCR neg-ative colostrum samples were spiked with M. haemolamae infected bloodas a means of determining detection limits of the assay in colostrum sam-ples and provide a relative detection limit to the same known positiveblood sample.

2.3. DNA sequencing

To evaluate the application of the M. haemolamae PCR test against ageographically diverse set of M. haemolamae isolates, M. haemolamae pos-itive alpaca blood from Ohio, Washington (WADDL, Pullman, WA, USA),Oklahoma (Shawnee Animal Hospital, Shawnee, OK, USA) and Georgia(Dr. Alessandra Pellegrini-Masini University of Georgia, Athens, GA, USA)were evaluated with primer set III (Table 1). M. haemolamae was ampli-fied (696 bp), and DNA sequencing of this 696 bp product was performedto target a region of DNA encompassing the targeted region of Primerset II. DNA sequence analysis of DNA from these amplified samples wereconducted using Vector NTI (version 11) (Vector NTI version 11, Invit-rogen/Life Technologies, CA, USA) and compared to sequences availablethrough the National Library of Medicine, GenBank website by BLASTanalysis (Altschul et al., 1990).

These directly sequenced (bidirectional) DNA fragments weredeposited in GenBank under the following accession numbers: JN214356(Oregon); JN214357 (Georgia); JN214358 (Okahoma); FJ527244 (Ohio,clinical case); and JN214359 (Ohio, in utero case). The alpaca sequenceproduced using primer set I is reported as GenBank accession JN214360.

2.4. Immunoglobulin testing

A Camelid Serum/Plasma and colostral IgG turbidimetric assay(Value Diagnostics, Turbidometric Immunoassay, Camelid IgG (blood,Colostrum), WI, USA) was used to determine serum IgG concentrationsin pre- and post-colostrum serum samples (cria), and colostrum sam-ples (dam). Serum IgG concentrations were determined for each cria aftercolostrum ingestion at the 72 h time point as part of this farm’s routinepreventive medicine program.

To prepare M. haemolamae antigen slides for indirect fluorescent anti-body testing, blood from a 3 month-old infected female alpaca cria wasused, containing the 0.25–0.5 �m, basophilic, non-refractile round bodiespresent individually and in rows on the surface of red blood cells (con-sistent with the appearance of a hemoplasma) (Reagan et al., 1990). Theblood was confirmed to be positive for M. haemolamae by PCR. This crias’

M. haemolamae-specific antibodies present in colostrum were evalu-ated by indirect fluorescent antibody test (IFAT). The IFAT was performedusing antigen prepared slides from red blood cells infected withM. haemolamae from the naturally infected blood smear positive cria

Primer sequence

5′-ATGCTTTTCTGTGTATGGTTATCTAGTG-3′

5′-CTAGTTTCCCAAGTTCATCTTTCTG-3′

5′-ACG AGC AGT GAG GAA TTT TTC AC-3′

5′-TCA ATT ATG TCC CAG GTA CTC-3′

5′-ACG AGC AGT GAG GAA TTT TTC AC-3′

5′-TGCACCACCTGTCATACCGATACC-3′

n control.unit ribosomal RNA gene.

184 R.L. Pentecost et al. / Small Ruminant Research 106 (2012) 181– 188

Table 2Number of Mycoplasma haemolamae PCR positive and PCR negative dams, PCR positive and negative cria immediately after birth, PCR positive and negativecolostrum samples and post-colostral testing of cria.

PCR results Dam Cria pre-colostrum Colostrum Cria post-colostrum

associated with PCR status of the cria at birth (Fisher’s exacttest; p = 0.3077). The pre-colostral, blood PCR positive cria,as well as all pre-colostral, blood PCR negative cria, were

Fig. 1. Ethidium bromide-stained agarose gel showing M. haemolamaespecific polymerase chain reaction (PCR) products of approximately 415

Positive 16 (30.8%) 1 (1.9%)

Negative 36 (69.2%) 51 (98.1%)

Total 52 52

described above. The infected red blood cells were placed onto multi-well slides (Cell Line Associates, NJ, USA) air-dried, fixed in methanol,and stored at −20 ◦C until used. Colostrum samples were assayed at dilu-tions of 1:10 and 1:100 in phosphate buffered saline (PBS). Briefly, 15 �l of1:10 and 1:100 dilutions of colostrum were added to each slide well, incu-bated in a humidified chamber at 37 ◦C for 45 min followed by three 5-minPBS washes. Ten microliters of fluorescein-labeled, affinity purified goatanti-llama IgG heavy and light chain (Bethyl Laboratories, anti-camelidIgG H + L; FITC conjugate, TX, USA) was diluted 1:200 in PBS and addedto each well, followed by incubation in a humidified chamber at 37 ◦Cfor 45 min. The slides were subjected to three, 5-min PBS washes, thencoverslipped with one drop of fluoromount G (Fluoromount G, SouthernBiotech, AL, USA). Slides were independently read by two individuals whowere blinded to specimen information (AEM, RLP) to verify the consis-tency of assay interpretation. The dam’s colostrum from the in utero M.haemolamae infected cria served as the positive control, while the diluent(1× PBS) served as the negative control.

2.5. Statistical analyses

Descriptive statistics were performed to determine the proportion ofthe PCR negative and positive dams and newborn cria. Fisher’s exact testwas used to evaluate whether PCR positive and negative status of thedams was associated with the likelihood of giving birth to a PCR positivecria (prior to ingestion of colostrum). In order to compare the presenceof M. haemolamae specific antibodies in colostrum with PCR status of thedam, a 2 × 2 contingency table was constructed to test the hypothesis thatPCR positive dams would have greater likelihood of having M. haemolamespecific antibodies than PCR negative dams. These data were analyzedby Chi-square test for independence. Colostral IgG concentrations amongdams of different ages (2–3 years, 4–5 years and older than 5 years) andbetween PCR positive and negative dams were compared using one-wayANOVA.

3. Results

3.1. PCR testing and DNA sequencing

Extracted DNA was successfully amplified from all indi-vidual samples included in the 4 sample sets analyzed foreach dam–cria pair using primer set 1, producing the pre-dicted 219-bp product. The DNA extracted from llama andcamel blood also produced a 219 bp amplicon using primerset I; however, this primer set failed to produce ampli-cons when primer set I was tested with DNA obtainedfrom equine, caprine, ovine, and bovine DNA. The 219 bpamplified alpaca sequence aligned to one contig of the2× Broad alpaca assembly, ARR0118852 with 96% identityover 193 bp.

Primer set II (M. haemolamae specific), amplified theexpected 415-bp product using DNA extracted from a bloodsmear-confirmed case of M. haemolamae, and no PCR prod-uct was identified when tested on DNA extracted fromwhole blood of a healthy adult alpaca that had tested neg-

0 043 (100%) 52 (100%)43 52

DNA from the other mammalian species tested. Primer setII also failed to produce an amplicon when tested against avariety of different mycoplasma species: M. haemofelis, M.haemoninutum, M. gallisepticum, M. bovis, and M. synoviae.

A 1:1000 dilution of the DNA extracted from the bloodof the positive control cria remained positive using primerset II but failed to amplify this product at a 1:10,000 dilu-tion, demonstrating the relative sensitivity of the assay ondiluted DNA extracted from blood. DNA extracted from theblood taken from the single cria with a pre-suckle positivePCR result remained positive when diluted 1:100. The PCRassay was also capable of detecting the presence of 1 �l ofinfected control blood when diluted 200 fold in a PCR neg-ative colostrum sample (1:200 dilution of sample prior toDNA extraction).

In a pilot study, the PCR prevalence in a non-breedingmale group housed on this farm (90 males; aged 1–7 years)was 35.5% (32/90 PCR positive; Lakritz et al., 2008, unpub-lished data). This study used primer set II and included apositive control (clinically affected cria) and negative con-trol (no DNA template in reaction mixture). Consideringboth the males sampled in a pilot study and those femalesincluded in this prospective study, the overall PCR preva-lence of M. haemolamae in this herd was 34% (48/142 testedby PCR of blood). The PCR prevalence in the females eval-uated in this study prior to parturition was 30.8% (16/52dams were PCR positive) and 36 of 52 dams were PCR neg-ative (69.2%) (Table 2).

One cria tested positive by PCR at parturition priorto colostrum ingestion (1.9%); whereas, 51 of 52 criawere negative by PCR prior to colostrum ingestion (98.1%)(Table 2). The dam of the single PCR positive cria (3 yearsold) was PCR negative for M. haemolamae from both bloodand colostrum. Overall, the PCR status of the dam was not

base pairs, as sized by molecular size markers. The samples in this geloriginated from the following animals: lanes 1, 4, 7: blood samples fromalpacas positive for M. haemolamae; lanes 2, 3, 5, 6, 8, 9, 10: blood samplesfrom alpacas negative for M. haemolamae; lane 11: PCR reaction negativecontrol; lane 12: PCR reaction positive control.

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lso PCR negative 72 h after ingesting colostrum. All 43 ofhe colostral samples were PCR negative for M. haemola-ae and the other Mycoplasma species parasites evaluated

n this study.

.2. Comparative 16S rDNA analysis of geographicallyiverse M. haemolamae

DNA sequences obtained from the naturally infectednimals in this study were compared to M. haemola-ae PCR positive samples obtained from different regions

f the United States. These DNA sequences alignedith published M. haemolamae sequences, with minor

equence variations from a single nucleotide differenceGenBank JF495171) to 16 nucleotide differences (Gen-ank FN908077). The 16S rDNA partial sequence from theaturally infected M. haemolamae blood-smear-positive

month old cria demonstrated 99% and 97% identity toenBank JF495171 and FN908077, respectively, with sev-ral other published M. haemolamae GenBank sequencesalling within this identity range. None of the polymor-hisms occurred in sequences corresponding to primer set

I’s annealing sites.

.3. Immunoglobulin testing

Mean serum, pre-colostrum IgG concentrations of the. haemolamae PCR negative cria were 68.7 ± 19 mg/dl

range from 23 to 111 mg/dl). Mean serum, post-colostrumgG concentrations of the M. haemolamae PCR negative cria

ere 1964 ± 813 mg/dl (range 120–2950 mg/dl (n = 51)).he pre-colostrum blood sample IgG concentration fromhe M. haemolamae positive cria was 415 mg/dl and follow-ng ingestion of colostrum was 2213 mg/dl. There were notatistical differences in mean post-colostral IgG achievedy the cria after ingestion of colostrum when comparingrias born to PCR positive or negative dams (p = 0.790).

Mean colostral IgG concentrations were 9310 mg/dlmedian: 9856 mg/dl; range: 168–24,000 mg/dl). When

ig. 2. Representative images of IFA slide wells showing the absence (left) and prncluded in this study. Colostral samples were diluted to 1:10 prior to placing

nti-llama-FITC conjugate antibody that was added to the slide wells to visualize

esearch 106 (2012) 181– 188 185

concentrations in dams >5 years old had mean concentra-tions of 10,232.4 ± 6455 mg/dl (Table 3). The differences inthe means of these 3 groups of females were not statisti-cally different (p = 0.600). The colostral IgG concentrationsin these 3 age groupings were not significantly different.Furthermore, when colostral IgG content was comparedby the PCR status of the dam mean colostrum IgG concen-trations in the PCR negative dams was 9517 ± 6000 mg/dl(range 168–24,000 mg/dl) and PCR positive dams was8730 ± 4610 mg/dl (range 608–15,000 mg/dl). These dif-ferences were not statistically significant (p = 0.790). Inaddition, when colostral IgG concentration was stratifiedby age and PCR status of the dams blood, there were nostatistical differences (ANOVA; p = 0.943) between age andPCR reaction using primer set 2.

M. haemolamae specific IgG was detected in 22 of 43(51%) of the colostrum samples by IFAT at 1:10 and in 12of 43 (28%) colostral samples at ≥1:100 (Fig. 2). Twenty-one of 43 colostrum samples tested negative by IFAT at1:10. Among the 14 PCR positive dams for which sufficientcolostrum was available to perform the IFAT, 7 (50%) werepositive for M. haemolamae antibodies at the 1:10 dilution.Of those 7 samples, only 4 had antibody present at 1:100.Of the 29 PCR negative dams for which sufficient colostrumwas available for testing, 15 (52%) had antibodies presentat 1:10. Of those 15 samples, 10 (66%) were positive at1:100 (Table 4). The pre-colostrum PCR positive cria’s titerwas 1:8 and the corresponding post-colostrum was 1:10.When comparing PCR status of dam’s blood to M. haemola-mae specific IgG in the colostrum, there was no relationshipbetween PCR status of the dam and M. haemolamae specificIgG (p = 0.9202).

4. Discussion

This study evaluated whether (1) M. haemolamae istransmitted in utero from dam to cria, (2) whether colostraltransfer of M. haemolamae occurs, and (3) whether colostralIgG and M. haemolame specific antibody are associated with

The herd we evaluated by PCR analysis of blood for M.haemolamae indicates the prevalence of M. haemolamaeis greater on some farms than that reported in previous

esence (right) of antibodies to M. haemolamae in colostrum from subjectsonto IFA slide. Antibody binding was detected using a secondary, goat

antibody binding to RBC parasites by fluorescence.

186 R.L. Pentecost et al. / Small Ruminant Research 106 (2012) 181– 188

Table 3Number and percentage of Mycoplasma haemolamae PCR positive and negative dams by age, colostral IgG concentrations by age and colostral M. haemolamaespecific IgG by IFAT (at 1:10 and 1:100 dilutions). Colostrum samples from 43/52 dams were available for analysis due to limited volume of colostrumprovided.

Age Number testingPCR positive

Colostrum IgGconcentration (mg/dl)

Colostral M.haemolamae IFA 1:10

Colostral M.haemolamae IFA 1:100

2–3 year 5/14 (35.7%) 8006 ± 3777

4–5 year 7/18 (38.9%) 8954 ± 4916

>5 year 4/20 (20.0%) 10,232 ± 6454

studies (30.8% PCR positive infection rate for herd females)(Kaufmann et al., 2010; Tornquist et al., 2010). Despitegreater M. haemolamae infection prevalence, only a sin-gle cria born during the study period was PCR-positiveprior to ingestion of colostrum. This is compatible withearlier reports demonstrating pre-partum transmission ofM. haemolamae DNA in blood (Tornquist et al., 2011). Ourresults suggest that the incidence of vertical transmissionin this herd is <2%. This differs from another recent studywhere 5 PCR positive females sampled pre-partum gavebirth to 2 PCR positive cria prior to ingestion of colostrum(Tornquist et al., 2011). Assuming that those 5 femalesreflect the herd incidence in that study, vertical transmis-sion could be more frequent on some farms. However, thatprior study did not report results of testing performed onPCR-negative pre-partum females in the herd, so true herdtransmission incidence may differ from that reported. Ourresults suggest that giving birth to a PCR positive cria is justas likely to be from PCR-negative dams as to PCR-positivedams.

The remaining 69% of the dams in this study were PCR-negative for M. haemolamae. In fact, the only PCR-positivecria born during our study came from this population ofPCR-negative females. The possibility of a spurious resultdue to amplicon contamination is possible, albeit, unlikelyas negative controls included during the analysis of thatsample were negative. This in utero infection, based upona positive PCR test of whole blood from the cria priorto ingestion of colostrum is also supported by elevatedpre-colostral serum IgG concentration (415 mg/dl). Whencompared to all other cria born on this farm during thestudy period (mean pre-colostral IgG concentrations were68.7 ± 19 mg/dl), the pre-colostral IgG concentration of theonly PCR positive cria was more than 2 standard deviations

Table 4Colostral antibody presence and absence in Mycoplasma haemolamae PCR positivM. haemolamae status, IFAT evaluation demonstrated 50% of the dams had colostr52% of the Mycoplasma haemolamae PCR negative dams possessed colostral IgG

when evaluated at a 1:100.

Dam Mycoplasma haemolamae PCR status Colostral antibody titer

1:10

Antibody present A

Positive (n = 14) 7 (50%)

Negative (n = 29) 15 (52%) 1Total (n = 43) 22 (51%) 2

7/11 (63.6%) 2/11 (18.2%)8/16 (50.0%) 6/16 (37.5%)7/16 (43.8%) 4/16 (25.0%)

negative cria with serum IgG concentrations <100 mg/dl,the elevated pre-colostral serum IgG concentrations inthe PCR positive cria suggests an in utero response toan antigen. While not determined, we presume the PCRpositive cria mounted immune responses to a M. haemola-mae antigen in utero. Transplacental transmission of otherpathogens affecting cattle (Neospora caninum, BVDV) andhumans (Toxoplasma gondii) are well documented and areassociated with detectable immune responses of the fetus(Trees and Williams, 2005; Williams et al., 2009; Robert-Gangneux et al., 2009; Singh, 2003).

Transplacental transmission is of special concern sincethe newborn cria, infected in utero could be at greater riskof clinical disease. Furthermore, this cria could serve as areservoir of infection to herdmates. While these possibili-ties are a concern, our data suggest other factors, such ascolostral IgG may be critical determinants of developingclinical disease.

In our study population colostrum was found to be freeof M. haemolamae when tested using PCR, suggesting thattransmission through colostrum is unlikely. These find-ings are consistent with a prior report, in which colostrumtested PCR negative although derived from dams (n = 5)with PCR positive blood (Tornquist et al., 2011). A possi-bility still exists that organisms may be transmitted fromthe dam via subclinical hemorrhage into mammary secre-tions post-partum, whether or not blood in colostrum isgrossly visible.

Furthermore, our data suggest a role for colostral anti-body to M. haemolamae in limiting infection in the cria. Inour study, the M. haemolamae PCR-positive cria (in uteroinfection) tested negative by PCR to M. haemolamae 72 hafter ingesting colostrum. This observation suggests clear-ance of the organism from the blood, at least to levels

e and negative dams included in this study. When grouped according toal IgG titers at 1:10, however, decreased to 29% at 1:100. Likewise, while

specific for M haemolamae at 1:10 dilution, this number declined to 34%

1:100

ntibody absent Antibody present Antibody absent

7 (50%) 4 (29%) 10 (71%)4 (48%) 8 (28%) 21 (72%)1 (49%) 12 (28%) 31 (72%)

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olostral IgG is present and could ostensibly function toeduce the number of parasites below the threshold ofetection by PCR. Our indirect fluorescence test (IFAT) uti-

ized M. haemolamae parasitized red blood cells from aaturally infected cria to determine whether an IgG compo-ent of colostral antibodies was specific for this organism.ur IFAT results demonstrate that M. haemolamae spe-ific antibodies are present within colostrum from bothCR-positive and -negative dams. The results of this assayuggest that maternal factors (such as IgG) could modu-ate parasitemia in the cria. These findings are supported byrior work that demonstrated M. haemolamae specific anti-odies (Eperythrozoon weyonii at that time) in the serum of

large number of camelids from a broad geographic areaMcLaughlin et al., 1990). The maternal IgG detected in thistudy, could protect the cria by aiding removal of thesearasites (e.g. by opsonization), thereby limiting clinicalisease. Information defining the impacts of M. haemolmaepecific antibodies could prove useful in understandingoth the transmission of M. haemolamae and the clinicalignificance of M. haemolamae infection (McLaughlin et al.,990; Tornquist et al., 2011).

Our results also demonstrated that the presence of M.aemolamae specific antibodies is not correlated to PCRased testing results. This suggests a component of theam’s immune system is capable of reducing the num-er of parasites to below the threshold for detection, whileroducing antibodies specific to this organism. This is alsoompatible with prior reports (Tornquist et al., 2009, 2011).t is therefore possible that prior infection may provide themmunologic mechanisms necessary to clear infection androtect the newborn that acquires the organism early in lifehile colostral antibodies are still in circulation. In addi-

ion, since colostral M. haemolamae has not been detected,pecial management practices such as heat-treatment orasteurization of colostrum are not likely to result in reduc-ions in M. haemolamae transmission to the cria.

Thus, the PCR based test does not provide suffi-ient information regarding the host-parasite interaction;pecifically the risk of infection or the outcomes of theeonate that acquires infection. While we recruited a herdith what we proposed would have suitable infectionrevalence, we only detected a single vertical transmissionvent. In fact, based upon our pilot PCR study, a sampleize of 48 was predicted to be sufficient to detect differ-nces in transmission from dam to cria. From this study,CR-prevalence in the breeding animals in this herd is0.7%. However, antibody prevalence in colostrum fromCR-positive and -negative dam’s appears to be as high 50%.

The presence of antibody specific to M. haemolamae mayxplain how clinical disease in neonatal cria could be pre-ented. Specific antibodies to M. haemolamae were noted in0% of all evaluated colostrum samples indicating immu-ity to disease could be passed to these neonates. Specificntibodies were present in 50% of the dams regardless ofheir PCR test status. The presence of antibody in both PCRositive and negative dams indicates that infection may be

esearch 106 (2012) 181– 188 187

haemolamae immunoglobulin in these animals, challengestudies would be required.

While this IFAT was capable of detecting M. haemolamaereacting antibodies in plasma and colostrum, the plasmasamples showed an increased level of non-specific fluores-cence making the results difficult to interpret. Therefore,evaluation of titered dilutions of the plasma samples wasnot pursued. The colostrum samples were screened at 1:10and 1:100 because of the limited availability of antigenslides. The dam’s colostrum of the in utero infected cria waspositive at 1:100 but negative at 1:500 thereby setting ourscreening dilutions.

IFAT is most frequently used to monitor seroprevalenceof disease through the identification of specific antibod-ies to a given organism (Ruegg et al., 2007). This testingmodality identifies individuals that have previously beeninfected with and mounted an immune response to a givenpathogen but does not necessarily identify those that arecurrently infected (McLaughlin et al., 1990; Ruegg et al.,2007). As such, IFAT status is unlikely to be rewarding asa primary testing method for determining positive or neg-ative infection status with M. haemolamae, or as a meansof determining the likelihood that a dam’s offspring willacquire infection or immunity.

It would be useful to follow groups of cria throughtheir first year to determine whether any test negative criabecame positive to M. haemolamae by PCR as well as tofollow the status of an in utero-infected cria later in life.Furthermore, it would also be important to determine ifsubsequent vertical transmission occurs.

Mode of transmission is a key aspect to understandingherd infectious disease status. Further studies are needed todefine the life cycle of this parasite, including the identifica-tion of biological and mechanical vectors. A more thoroughunderstanding of the life-cycle and modes of transmissionwould provide information that may be useful in the devel-opment of strategies to prevent and manage this disease.Nonetheless, our study confirms the potential for in uterotransmission while showing that colostral transmission isunlikely for infection of cria by M. haemolamae. Colostraltransfer of M. haemolamae specific antibodies may play arole in preventing infection in the neonate.

Acknowledgements

Supported by the Morris Animal Foundation and theAlpaca Research Foundation. The authors thank Heather-brook Alpaca Farm and Bernie Younkman for technicalassistance, and Dr. Joan Pontius of the Laboratory ofGenomic Diversity, NIH for alpaca comparative genomicanalysis.

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