Veterinary Microbiology 149 (2011) 262–268

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Veterinary Microbiology

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Short communication

Novel hemotrophic mycoplasma identified in naturally infectedCalifornia sea lions (Zalophus californianus)

Dmitriy V. Volokhov a,*, Tenaya Norris b, Carlos Rios b, Maureen K. Davidson c,Joanne B. Messick d, Frances M. Gulland b, Vladimir E. Chizhikov a

a Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM-470, Rockville, MD 20852, United Statesb The Marine Mammal Center, Marin Headlands, 2000 Bunker Road, Fort Cronkhite, Sausalito, CA 94965, United Statesc Center for Veterinary Medicine, Food and Drug Administration, 8401 Muirkirk Road, Laurel, MD 20708, United Statesd Department of Comparative Pathobiology, Purdue University, School of Veterinary Medicine, 725 Harrison Street, West Lafayette, IN 47907, United States

A R T I C L E I N F O

Article history:

Received 27 September 2010

Received in revised form 17 October 2010

Accepted 29 October 2010

Keywords:

Hemoplasma

Blood

California sea lions

A B S T R A C T

The hemoplasmas are the trivial name for a group of erythrocyte-parasitizing, non-

cultivable in vitro bacteria of the genus Mycoplasma that have been described in several

mammalian hosts worldwide. This study is the first report of hemoplasmas in marine

mammals. EDTA anticoagulated whole blood samples from 137 California sea lions

(Zalophus californianus) and 20 northern elephant seals (Mirounga angustirostris) admitted

to the Marine Mammal Center (Sausalito, CA; www.marinemammalcenter.org) or live

captured in Oregon were collected during 2008. Hemoplasma-specific genomic DNA was

detected in blood samples from 12.4% California sea lions tested using PCR. Hemoplasma

PCR positive blood specimens also were tested in reverse transcription polymerase chain

reaction (RT-PCR) using the hemoplasma-specific primers for the 16S and 23S rRNA genes.

The RT-PCR showed the presence the hemoplasmal rRNA, strongly suggesting the

presence of potentially viable hemoplasmas in the bloodstream of the animals. BLAST

search and phylogenetic analysis of the 16S rRNA sequences of the hemoplasma from

California sea lions revealed that the organism is a novel hemoplasma species with only

92.1% of its nucleotide similarity to the 16S rRNA gene of the previously described

hemoplasma species of alpacas, Candidatus Mycoplasma haemolamae. Thus, due to low

level of genetic similarity of the hemoplasma to other described hemoplasmas and the

mammalian host in which the hemoplasma was detected we propose that this novel

hemoplasma species has been given the provisional name Candidatus Mycoplasma

haemozalophi sp. nov.

Published by Elsevier B.V.

1. Introduction

The hemoplasmas (the common name for hemotrophicMycoplasma species) are facultative intracellular, cell wall-less, erythrocytic parasites comprising the group of non-

* Corresponding author at: U.S. Food and Drug Administration, Center

for Biologics Evaluation and Research, Office of Vaccine Research and

Review, Division of Viral Products, Laboratory of Methods Development,

HFM-470, 1401 Rockville Pike, Rockville, MD 20852, United States.

Tel.: +1 301 827 8757; fax: +1 301 827 9531.

E-mail address: dmitriy.volokhov@fda.hhs.gov (D.V. Volokhov).

0378-1135/$ – see front matter . Published by Elsevier B.V.

doi:10.1016/j.vetmic.2010.10.026

cultivable Mycoplasma species that include those organismsformerly known as Haemobartonella and Eperythrozoon

species, which were reclassified as Mycoplasma speciesbased on the phylogenetic analysis of their 16S rRNA genesequences and deeper studies on cell morphologic proper-ties (Messick, 2004; Messick et al., 2002; Neimark et al.,2001, 2002; Willi et al., 2007). Hemoplasmas are thecausative agents of acute or chronic infectious anemias innumerous mammalian species (Messick, 2004). Humaninfections with hemotrophic mycoplasmas also have beenreported (dos Santos et al., 2008; Sykes et al., 2010). Themain hematological observation in hemoplasma-infected

D.V. Volokhov et al. / Veterinary Microbiology 149 (2011) 262–268 263

animals is mild or severe anemia (Messick, 2004; Stoffregenet al., 2006), but infections can occur with or withouthematological signs of anemia (Almy et al., 2006; Groebelet al., 2009; Hoelzle et al., 2009; Messick, 2004; Ritzmannet al., 2009; Stoffregen et al., 2006; Willi et al., 2007). Theanemic illness can be more severe in young, splenectomized,and immunocompromised animals (Messick, 2004). Hemo-trophic mycoplasmas can be found as free-floating bacterialcells or attached to the surface of host red blood cells andthey are able to invade erythrocytes under some clinicalcircumstances (Groebel et al., 2009; Willi et al., 2007). Theintracellular invasion of host erythrocytes by the hemo-plasmas is proposed as a mechanism to escape detection andkilling by innate and immune mediated host defenses aswell as killing by antibiotics that have less membranepenetrability (Groebel et al., 2009; Vogl et al., 2008). Theintracellular life cycle of some hemotrophic mycoplasmasalso may explain the chronic nature of hemotrophicmycoplasma infections (Groebel et al., 2009).

The goal of the present study was to determine theexistence of hemotrophic mycoplasmas in two wild caughtmarine mammalian species, California sea lions (CSL;Zalophus californianus) and northern elephant seals (Mir-

ounga angustirostris), in California and Oregon.

2. Materials and methods

2.1. Sample collection and medical records

EDTA-anticoagulated blood samples were collectedfrom 137 California sea lions and 20 northern elephantseals that were admitted to the Marine Mammal Centerfacilities in California or captured for sampling in Oregon,USA. The samples were collected and frozen at�80 8C untiltheir analysis. The following information was collected forall the tested animals: species, age, gender, clinical status,and diagnosis. Blood samples were collected from eachanimal for a complete blood count (CBC; Vet ABChematology analyzer, Heska Corporation, Fort Collins,CO, USA), a manual 200-cell differential count, and aclinical chemistry profile (Olympus AU5200 ChemistryImmuno Analyzer, Olympus America Inc., Melville, NY,USA) within 48 h of admission.

2.2. Nucleic acid preparations

Total DNA and RNA were extracted from 200 ml ofEDTA-anticoagulated blood using DNeasy blood and tissuekit and RNeasy Protect Mini Kit, respectively (QIAgen,Chatsworth, CA) according to the manufacturer’s proto-cols. DNA and RNA were stored at �80 8C until use.

2.3. PCR amplification

The fragments of the16S and 23S RNA genes wereamplified from blood DNA samples using the PCR primersdesigned for this study (Table S1). Briefly, the standard PCRmixture (50 ml) contained 1.5 U of HotStar Taq DNApolymerase, 1x reaction buffer supplemented with2.5 mM MgCl2 (Qiagen, Chatsworth, CA), 500 nM of eachforward and reverse primer, a 200 mM concentration of

each deoxyribonucleoside triphosphate (dATP, dGTP, dCTP,and dTTP), and 5–7 ml of DNA template (ca. 0.1–0.2 mg oftotal DNA). The PCR was performed using a GeneAmp PCRsystem model 9700 thermocycler (PE Applied Biosystems,Foster City, CA), with the following cycle conditions: initialactivation at 95 8C for 15 min; 40 cycles of 94 8C for 1 min,56 8C for 1 min, and 72 8C (extension) for 2 min; and a finalextension at 72 8C for 7 min. The synthesis of PCR productswith the expected molecular weights was confirmed byelectrophoresis using a 1% SeaKem Gold TAE-agarose gelwith ethidium bromide (Lonza, Allendale, NJ) followed byUV visualization. The sizes of the 16S and 23S RNAamplicons were 1438 bp and 1795 bp, respectively. Theamplicons obtained were directly sequenced. The absenceof PCR inhibitors in isolated blood DNAs was monitored byPCR amplification of the mammalian mitochondrial 16SrRNA gene as described elsewhere (Volokhov et al., 2008).

2.4. Detection of the 16S rRNA in blood samples using RT-PCR

amplification

The presence of the hemoplasmal 16S and 23S rRNA inthe total RNA samples isolated from the blood specimenswas confirmed by using one-step reverse transcriptionpolymerase chain reaction (RT-PCR) with the PCR primersspecifically designed for this novel hemotrophic Myco-

plasma species (Table S1). To remove any traces ofhemoplasmal DNA in the RNA samples the latter weretreated with TURBO DNase (Ambion, CA) in accordancewith the manufacturer’s protocol. After the treatment, theTURBO DNase in the samples was inactivated by heating at75 8C for 15 min. The blood RNA samples shown to be freeof hemoplasmal genomic DNA by PCR with the 16S and 23SrRNA gene-specific primers were used for the detection ofrRNA transcripts of these genes by using RT-PCR. Briefly,QIAGEN OneStep RT-PCR Kit, 500 nM of each forward andreverse primer, and 7 ml of extracted RNA template wereused in this reaction. The RT-PCR was performed using aGeneAmp PCR system model 9700 thermocycler (PEApplied Biosystems, Foster City, CA) with the followingcycle conditions: RT-step at 57 8C for 60 min; initialactivation at 95 8C for 15 min; 40 cycles of 94 8C for1 min, 58 8C for 1 min, and 72 8C (extension) for 1 min; anda final extension at 72 8C for 7 min. The synthesis of PCRproducts with the expected molecular weights wasconfirmed by electrophoresis using a 1% SeaKem GoldTAE-agarose gel with ethidium bromide (Lonza, Allendale,NJ) followed by UV visualization. The sizes of the 16S and23S rRNA-specific amplicons were 636 bp and 1004 bp,respectively.

2.5. Phylogenetic analysis of sequences and construction of

phylogenetic trees

The obtained sequences of the hemoplasmal 16S and23S rRNA genes were compared to those available in theGenBank nucleotide database using procedures andalgorithms as described elsewhere (Volokhov et al.,2007). The 23S rRNA gene sequences of several Myco-

plasma and Ureaplasma species that were unavailable inthe GenBank were determined during the study. The 23S

D.V. Volokhov et al. / Veterinary Microbiology 149 (2011) 262–268264

rRNA gene was amplified with the universal primers(Table S1) under the PCR conditions described above.

2.6. Nucleotide sequence accession numbers

DNA sequences from this study were deposited inGenBank under accession numbers GU124600–GU124614,GU904996–GU905012, GU905033, HM135458–HM138663, HM135465–HM135467, HM197715, HM197716, andHM241735.

3. Results

3.1. Blood sample characteristics

Overall, 17/137 (12.4%) CSL and 0/20 northern elephantseals had evidence of hemoplasma infection. A total of 149of the 157 tested animals were diseased and theirindividual diagnoses included either one pathologicalcondition (i.e. malnutrition, local or systemic infections,traumas, leptospirosis, domoic acid intoxication, andlungworm infestation) or their combinations. Most ani-mals were males (70%) with yearlings and juveniles aspredominant age groups. There were no significantdeviations from normal hematological ranges in thehematological parameters for either species. Also, nocharacteristic hemoplasma-like organisms were observedon the May-Gruenwald–Giemsa stained blood smears. Thepresence or absence of hemoplasmas in all tested animalswere confirmed by PCR and sequencing. A summary of thehemoplasma-positive animals is presented in Table 1.Most of the CSLs (132/137; 96.3%) tested and all animalswith hemoplasmosis also were infected with a filarialnematode, Acanthocheilonema species, of the familyOnchocercidae. Microfilariae were detected on May-Gruenwald–Giemsa stained blood smears and weregenetically identified to the genus level by combinationof PCR amplification and sequencing of the mitochondrialand genomic regions suitable for the identification(Casiraghi et al., 2004). Based on the sequence similarity

Table 1

Summary about hemoplasma-positive California sea lions.

Animal no. Age group Sex Strand locality

CSL-7666 Adult Female Oceano Dunes, San Luis Obispo C

CSL-7750 Adult Female San Tomas Aquinas Creek, Santa

CSL-7755 Adult Female Oceano Dunes State Vehicular Re

San Luis Obispo Co.

CSL-7758 Adult Female Pismo State Beach, San Luis Obisp

CSL-7801 Juvenile Male Coast Guard Jetty, Monterey Co.

CSL-7783 Yearling Male Rio Del Mar Beach, Santa Cruz Co

CSL-7822 Juvenile Male Pier 41, San Francisco Co.

CSL-7860 Juvenile Male Middle Harbor Shoreline Park, Ala

CSL-7871 Adult Female Asilomar State Beach, Monterey C

CSL-7873 Juvenile Male Breakwater Cove Marina, Monter

CSL-7881 Juvenile Male Ocean Beach, San Francisco Co.

CSL-7897 Juvenile Male Dunes Beach, San Mateo Co.

C606 Juvenile Male Bonneville Dam, Oregon

C795 Juvenile Male Bonneville Dam, Oregon

C797 Juvenile Male Bonneville Dam, Oregon

C796 Juvenile Male Bonneville Dam, Oregon

C739 Juvenile Male Bonneville Dam, Oregon

Note: All these hemoplasma-positive California sea lions also were infected with a

and phylogenetic analysis of the 18S small subunit rRNA,coI (cytochrome oxidase I), and sod1 (putative copper/zincsuperoxide dismutase) genes, the filarial nematode specieswas found to be closely related to Acanthocheilonema viteae

with 99%, 89% and 91% nucleotide similarity, respectively.

3.2. PCR primers design and PCR amplification of

hemoplasma-specific sequences

To assess the prevalence of hemoplasmal bacteremia inthe blood samples of the marine mammals, special PCRprimers were designed based on the conserved sequencesfound within the 16S rRNA gene sequences of all knownhemotrophic mycoplasmas and phylogenetically closelyrelated Mycoplasma and Ureaplasma species (Fig. S1). The16S rRNA phylogenetic tree had two well divided clusterswith bootstrap value of 98% for each cluster. The cluster Iincluded all known hemotrophic Mycoplasma speciestogether with three non-hemotrophic species, M. cavi-

pharyngis, M. fastidiosum and M. insons, while the cluster IIcomprised other non-hemotrophic Mycoplasma and Urea-

plasma species. Based on the phylogenetic analysis and the16S rRNA gene sequences of Mycoplasma species withinthe cluster I, we designed the PCR primers for screening ofall blood specimens that matched most conserved regionsof all hemotrophic Mycoplasma and non-hemotrophic,closely related Mycoplasma species (Table S1). We alsoused other PCR primers, 8F and 1492R, which werepreviously demonstrated to be suitable for the PCR-baseddetection of hemotrophic Mycoplasma ovis (Neimark et al.,2004). The evaluation of the newly designed, broadlyspecific primers, as well as the previously publishedprimers, showed that the primers were able to amplify the16S rRNA sequences of all non-hemotrophic closely relatedMycoplasma and Ureaplasma species. However, theseprimers also amplified the 16S rRNA gene sequences ofStreptococcus phocae, Staphylococcus warneri and Salmo-

nella enterica subsp. enterica, which also were found in twohemoplasma-positive and several hemoplasma-negativeblood samples. Only one hemoplasma-specific sequence

Diagnosis Outcome

o. Chronic domoic acid toxicity Euthanized

Clara Co. Chronic domoic acid toxicity Euthanized

creation Area, Trauma, carcinoma Euthanized

o Co. Leptospirosis Released

Leptospirosis, pneumonia Euthanized

. Malnutrition, lungworms Released

Leptospirosis Euthanized

meda Co. Leptospirosis Released

o. Chronic domoic acid toxicity Euthanized

ey Co. Leptospirosis, septicemia Euthanized

Leptospirosis Died in treatment

Leptospirosis Released

Healthy Released

Healthy Released

Healthy Released

Healthy Released

Healthy Released

filarial nematode, Acanthocheilonema species, of the family Onchocercidae.

D.V. Volokhov et al. / Veterinary Microbiology 149 (2011) 262–268 265

with minor single point mutations (�1%) was detectedamong the sequences obtained from CSL hemoplasma-positive blood samples. To enable the specific PCR-baseddetection of novel hemoplasma species, new sets of PCRprimers were designed using unique sequences foundwithin the 16S rRNA and 23S rRNA genes of the CSLhemoplasma (Table S1). All blood samples were retestedwith these new primers. The new primers specificallyamplified only the novel hemoplasma species but not anyother bacterial microorganisms previously detected in thesamples using the broadly specific primers. We suggestemploying these specific primers for diagnostic PCRscreening of California sea lions blood samples for thepresence of this particular hemoplasma species.[()TD$FIG]

Fig. 1. Dendrogram showing phylogenetic relationships based on nucleotide seq

(the provisional name Candidatus M. haemozalophi sp. nov.) with other hemotrop

related Mycoplasma spp. (M. insons, M. fastidiosum, and M. cavipharyngis). The tr

package. The images are representing mammalian and insect species in which

To determine whether the hemoplasma were viableorganisms in the blood of the sea lions, we tested the bloodspecimens of PCR positive animals by RT-PCR usingthe hemoplasma-specific 16S and 23S rRNA primers(Table S1). The presence the hemoplasmal rRNA detectedin the CSL blood strongly indicated the viability of themicroorganisms.

To amplify the 23S rRNA gene of this novel hemo-plasma species from the blood samples, which were thehemoplasma-positive in the 16S rRNA-based PCR, wedesigned a set of universal primers (Table S1). At the timethis study was initiated, there were no 23S rRNAsequences available for all known hemotrophic Myco-

plasma species. Thus, the universal primers were designed

uence data for the 16S rRNA gene among the novel hemoplasma species

hic Mycoplasma spp., and three non-hemotrophic phylogenetically closely

ees were constructed by the minimum evolution method in the MEGA 4

those hemoplasma species were detected.

D.V. Volokhov et al. / Veterinary Microbiology 149 (2011) 262–268266

based on the 23S rRNA sequences available for non-hemotrophic Mycoplasma and Ureaplasma species (M.

pneumoniae, M. genitalium, M. gallisepticum, M. penetrans,and U. urealyticum) known to be phylogenetically closelyrelated to known hemoplasmas (Fig. S1). PCR products fromall hemoplasma-positive blood samples were amplifiedusing the designed 23S rRNA universal primers andsequenced. No PCR amplification was observed whenhemoplasma-negative blood samples were tested. Negli-gible intragenic variations (�1%) among the 23S rRNA genesequences of this novel hemotrophic mycoplasma detectedin the different animals were observed. All our attempts toamplify the 16S–23S internal transcribed spacer region (ITS)from all the hemoplasma-positive blood specimens wereunsuccessful. The separate localization of the 16S and 23SrRNA genes in the genome of M. suis, which is phylogen-etically closely related to the CSL hemoplasma, has beennoted by Dr. Messick (personal communication).

3.3. Phylogenetic analysis of the 16S rRNA and the 23S rRNA

genes

We used the sequences of the16S and 23S rRNA genesto determine the phylogenetic relatedness of the novelhemoplasma with other hemotrophic Mycoplasma andnon-hemotrophic, closely related, Mycoplasma and Urea-

[()TD$FIG]

100

100

99

100

85

100

95

100

100

100

98

100

100

77

88

0.020.040.060.080.100.120.14

Fig. 2. Dendrogram showing phylogenetic relationships based on nucleotide sequ

provisional name Candidatus M. haemozalophi sp. nov.) with other hemotrophic M

Mycoplasma spp. (M. insons, M. fastidiosum, and M. cavipharyngis). The trees were

plasma species (Figs. 1 and 2). The dendrograms in Figs. 1and 2 showed the inferred phylogenetic position of ournew hemoplasma species among known hemotrophicMycoplasma spp. and three non-hemotrophic but closelyrelated mycoplasmas (M. insons, M. fastidiosum, and M.

cavipharyngis). The interspecies similarity of the 16S rRNAand the 23S rRNA genes among species was assessed(Tables S1 and S2). The 16S rRNA-based phylogeneticanalysis demonstrated that the novel hemoplasma speciesfrom California sea lions was phylogenetically most closelyrelated to known hemoplasma species, Candidatus Myco-

plasma haemolamae, which was previously described inalpacas (Lama pacos) (Almy et al., 2006; Lascola et al., 2009;Messick et al., 2002). The 23S rRNA-based phylogeneticanalysis demonstrated that the novel hemoplasma speciesfrom California sea lions formed a separate branch withtwo other hemoplasmas, M. haemofelis and M. suis, with M.

suis being most closely related to the CSL hemoplasma.There was no 23S rRNA gene sequence available for thealpacas’ hemoplasma, M. haemolamae, and therefore thesimilarity between the 23S rRNA gene sequences of the CSLhemoplasma and the alpacas’ hemoplasma cannot beassessed. Based on the position on the phylogenetic tree(Fig. 2) and nucleotide similarity data for the 23S rRNAgene (Table S2), M. suis was found to be the geneticallyclosest species to the CSL hemoplasma.

AF222894 U. parvum

CP001184 U. urealyticum

HM197715 U. cati

HM197716 U. felinum

U. canigenitalium

HM135466 M. microti

L43967 M. genitalium

U00089 M. pneumoniae

AE015450 M. gallisepticum

HM135465 M. alvi

GU905033 M. amphoriforme

HM138662 M. testudinis

HM135467 M. insons

HM135458 M. fastidiosum

HM138663 M. cavipharyngis

M. haemofelis

GU905005 Candidatus M. haemozalophi sp. nov.

M. suis

0.00

ence data for the 23S rRNA gene among the novel hemoplasma species (the

ycoplasma spp., and three non-hemotrophic phylogenetically closely related

constructed by the minimum evolution method in the MEGA 4 package.

D.V. Volokhov et al. / Veterinary Microbiology 149 (2011) 262–268 267

4. Discussion

The genus Mycoplasma comprises several hemotrophicmycoplasma species which infect different hosts andattach to, and sometime invade, their erythrocytes(Groebel et al., 2009; Willi et al., 2007). Hemoplasmashave not been cultured in vitro and detection ofhemotrophic mycoplasmas using Romanowsky–Giemsatype stained blood smears in combination with specificPCR remains the gold standard for detection of theseinfections in animals and humans (Almy et al., 2006; dosSantos et al., 2008; Willi et al., 2007). Remarkably, theprevalence of hemoplasma-infected animals in hostpopulations has been reported to be around 0.5–15.4%(Hackett et al., 2006; Hoelzle et al., 2009; Kenny et al.,2004; Roura et al., 2010; Sykes et al., 2008). The infection ofanimals with hemotrophic mycoplasmas is usually self-limited and the establishment of inapparent chronicbacteremia is possible in immunosuppressed or immuno-compromised individuals (dos Santos et al., 2008; Yuanet al., 2007).

This study represents the first report of a hemotrophicMycoplasma species in marine mammals. Although about13% of CSL were infected with hemoplasma in this study,the majority of the studied animals were diseased (149/154; 96.7%) and thus no estimate of prevalence of theinfection in clinically normal animals can be made. Allblood samples from 20 northern elephant seals tested werehemoplasma-negative, but since only a few animals wereexamined, hemoplasma prevalence data cannot be deter-mined for this species. Assuming that the lowest pre-valence of hemoplasma infection in marine mammals issimilar to the 0.2–0.5% reported for some other hostspecies (Roura et al., 2010; Willi et al., 2006), a minimum as100–200 animals are required to confidently detect theexistence the infection.

The sequencing analysis of the hemoplasma-specific16S and 23S amplicons from the CSL blood samplesrevealed only minor single nucleotides variations (�1%).Thus, the obtained sequence data indicate the existence ofone hemoplasma species in CSL populations inhabitingCalifornia and Oregon.

The infections of animals with hemotrophic Myco-

plasma species are often associated with different types ofanemias and positive Coombs tests (Messick, 2004;Tagawa et al., 2010; Willi et al., 2007). However, thelaboratory or clinical manifestations of anemia andalteration of other hematological parameters do notalways accompany the hemoplasmal infection. In ourcases, the presence of the hemotrophic mycoplasmaorganisms in CSLs was not accompanied by anemia, eventhough all hemoplasma-positive animals also were simul-taneously infected with filarial nematodes of the genusAcanthocheilonema.

Others have stated that subjective evaluations ofhemoplasma infections may lead one to the conclusionthat the numbers of hemoplasma present in the bloodseemed to be correlated with the level of anemia in hosts(Willi et al., 2007). Our observation on the absence ofanemia in hemoplasma-positive CSLs may reflect a lowpathogenicity of this hemoplasma species for the host or

adaptation in a host-parasite system (Schulte et al., 2010).Because we have detected hemoplasmas in CSLs ofdifferent age groups (adults, yearlings, and juveniles),the affect of hemoplasmosis on the health of differentindividuals should be studied in the future.

Transmission of hemotrophic mycoplasmas amonganimals other than CSLs has been shown to be accom-plished by a number of blood-feeding arthropod vectors,including mosquitoes and stable flies, as well as directtransmission via infected blood (e.g. aggressive interac-tion, blood transfusion, and by an iatrogenic route) (Williet al., 2007). Potentially, all these routes of transmissionmay work for hemoplasma spreading in CSL populations.Pinnipeds are known to have sucking lice, Echinophthirius

horridus (Leidenberger et al., 2007). The seal louse issuggested to play an important role as an intermediatehost transmitting the heartworm Acanthocheilonema

spirocauda among seals (Leidenberger et al., 2007). Thepotential role of the seal louse in the transmission ofhemoplasmal infection within CSL populations is spec-ulative, but warrants additional investigation. Additionalroutes of transmission for hemoplasmas appear to exist aswell. For example, transplacental and transmammary(through colostrum) transmissions of M. haemolamae,which is closest phylogenetically related to our novelhemoplasma, have been suggested in alpacas (Almy et al.,2006). Clearly, additional study of the natural history ofhemoplasma infections in many species is warranted.

Finally, we propose that the newly discovered hemo-trophic Mycoplasma isolates from California sea lions (Z.

californianus) to be given the taxonomic status Candidatus

Mycoplasma haemozalophi. The Candidatus designation isappropriate because it is specifically reserved for newlydescribed, non-cultivable and incompletely characterizedspecies in order to give them provisional status (Brownet al., 2007; Murray and Schleifer, 1994; Murray andStackebrandt, 1995).

Acknowledgement

We express our appreciation to Dr. Victoria Majam forher help with staining of blood smears.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.vetmic.2010.

10.026.

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