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Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice

Published online by Cambridge University Press:  25 August 2015

J. P. DUBEY ,
S. K. VERMA ,
D. DUNAMS ,
R. CALERO-BERNAL  and
B. M. ROSENTHAL
J. P. DUBEY*
Affiliation:
U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
S. K. VERMA
Affiliation:
U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
D. DUNAMS
Affiliation:
U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
R. CALERO-BERNAL
Affiliation:
U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
B. M. ROSENTHAL
Affiliation:
U. S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA
*
*Corresponding author. USDA, ARS, APDL, BARC-East, Building 1001, Beltsville, MD 20705, USA. E-mail: jitender.dubey@ars.usda.gov
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Summary

The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50–220 days p.i. Sarcocysts contained unique villar protrusions, ‘type 38’. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri.

Keywords

Sarcocystis speeriSarcocystis falcatulaSarcocystis neuronaOpossum (Didelphis virginiana)Diagnosis
Type
Research Article
Information
Parasitology , Volume 142 , Issue 13 , November 2015 , pp. 1555 - 1562
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Copyright
Copyright © Cambridge University Press 2015 

INTRODUCTION

The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula (Box and Duszynski, Reference Box and Duszynski1978; Box et al. Reference Box, Meier and Smith1984), Sarcocystis neurona (Dubey et al. Reference Dubey, Davis, Speer, Bowman, de Lahunta, Granstrom, Topper, Hamir, Cummings and Suter1991; Fenger et al. Reference Fenger, Granstrom, Gajadhar, Williams, McCrillis, Stamper, Langemeier and Dubey1997; Dubey and Lindsay, Reference Dubey and Lindsay1998, Reference Dubey and Lindsay1999) and Sarcocystis speeri (Dubey et al. Reference Dubey, Speer and Lindsay1998; Dubey and Lindsay, Reference Dubey and Lindsay1999). It appears that there may be additional undescribed species of Sarcocystis in D. virginiana feces (Tanhauser et al. Reference Tanhauser, Yowell, Cutler, Greiner, MacKay and Dame1999; Dubey et al. Reference Dubey, Calero-Bernal, Rosenthal, Speer and Fayer2015). The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) act as the definitive host for S. falcatula and Sarcocystis lindsayi (Dubey et al. Reference Dubey, Venturini, Venturini, Basso and Unzaga1999, Reference Dubey, Kerber, Lindsay, Kasai and Pena2000a , Reference Dubey, Lindsay, Rezende and Costa b ; Reference Dubey, Rosenthal and Speer2001b ; Stabenow et al. Reference Stabenow, de Oliveira, Albuquerque and Lopes2008, Reference Stabenow, Ederli, Lopes and de Oliveira2012). Additionally, Didelphis albiventris is a definitive host for S. neurona (Dubey et al. Reference Dubey, Lindsay, Kerber, Kasai, Pena, Gennari, Kwok, Shen and Rosenthal2001a ). The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically, in part because of the non-availability of distinguishing genetic markers (Dame et al. Reference Dame, MacKay, Yowell, Cutler, Marsh and Greiner1995; Tanhauser et al. Reference Tanhauser, Yowell, Cutler, Greiner, MacKay and Dame1999).

Infectivity to immunodeficient mice and budgerigars (Melopsittacus undulatus) has been one important means to distinguish among species of Sarcocystis in opossum feces (Dubey, Reference Dubey2000). Sarcocystis neurona and S. speeri are infective to immunodeficient mice but not budgerigars, whereas S. falcatula and S. lindsayi are infectious for budgerigars, but not mice (Marsh et al. Reference Marsh, Barr, Tell, Koski, Greiner, Dame and Conrad1997; Dubey et al. Reference Dubey, Speer and Lindsay1998; Dubey and Lindsay, Reference Dubey and Lindsay1999). However, there is an unconfirmed report of cowbirds (Molothrus ater) as an intermediate host of S. neurona (Mansfield et al. Reference Mansfield, Mehler, Nelson, Elsheikha, Murphy, Knust, Tanhauser, Gearhart, Rossano, Bowman, Schott and Patterson2008). Both S. neurona and S. speeri can induce encephalitis in mice associated with schizonts and merozoites (Dubey et al. Reference Dubey, Speer and Lindsay1998; Dubey and Lindsay, Reference Dubey and Lindsay1999), but S. neurona does not form sarcocysts in mice (Dubey et al. Reference Dubey, Rosenthal and Speer2001b ). Sarcocystis speeri, however, can form sarcocysts in interferon gene knockout (KO) and nude mice, but full details of sarcocyst development and details of bradyzoite structure are not known (Table 1). Natural intermediate hosts for S. speeri, also remain unknown.

Table 1. Transmission electron microscopic examination of sarcocysts of Sarcocystis speeri

a Transmitted to Didelphis virginiana via KO mice from Didelphis albiventris.

Sarcocystis speeri has not been characterized molecularly because of the possibility of several species of Sarcocystis sporocysts in opossum feces. Unlike S. neurona, S. speeri was cultivated in vitro only once (Dubey et al. Reference Dubey, Speer and Lindsay2000d ). This culture was obtained from an Argentinian opossum, D. albiventris. Sarcocystis neurona, unlike S. speeri does not form sarcocysts in any strain of mice.

Here we report the development of S. speeri sarcocysts in KO mice. We also provide molecular characterization of S. speeri from culture derived merozoites with the objective that findings may be helpful in the discovery of its natural intermediate host.

MATERIALS AND METHODS

For the present study, Sarcocystis sporocysts were obtained from the intestinal scrapings of two adult opossums # 9062 and # 9068 (Table 1) using procedures described by Dubey (Reference Dubey2000). Opossum # 9062 was D. virginiana from Metro Zoo, Florida and received at the Animal Parasitic Diseases Laboratory (APDL) on 12 April 1999. It had a mixed infection of S. falcatula and S. speeri sporocysts in feces, based on bioassay in mice and budgerigars (Dubey, Reference Dubey2000). Sporocysts from opossum # 9062 were fed to KO mice and sporocysts had been stored at 4 °C for 30 days before inoculation of mice.

Sporocysts from opossum # 9068, D. albiventris were received at APDL from Argentina on 21 April 1999, and information on this specimen is reported here for the first time. Sporocysts from opossum # 9068 were inoculated orally into 18 KO mice (BALB/c-Ifngtm1Ts) that were euthanized 1, 2, 4 (2 mice), 6, 8, 9, 11 (2 mice), 14, 17, 22, 25, 46, 52, 59, 104 and 140 days post-inoculation (p.i.). The sporocysts had been stored at 4 °C for 28–70 days before oral inoculation into KO mice in 1999. Sporocysts from opossum # 9062 were inoculated orally into 16 KO mice, and the mice were euthanized on days 1, 3, 5, 7, 9, 11, 13, 16 (2 mice), 34, 35, 37, 38, 39 (3 mice); the sporocysts had been stored in refrigerator from 1 to 135 days. The KO mice were obtained from Jackson Laboratories as described by Dubey and Lindsay (Reference Dubey and Lindsay1998).

The number of S. speeri sporocysts in feces of the opossums was unknown; both opossums had also S. falcatula sporocysts as revealed by bioassay in budgerigars (data not shown). For estimation of S. speeri sporocysts in opossum # 9068, 10-fold serial dilutions were inoculated orally into groups of two KO mice. These inoculated mice were observed for 83 days. Bradyzoites were collected by pepsin digestion of infected muscles as described (Dubey et al. Reference Dubey, Speer and Fayer1989).

Mice that were killed or died were necropsied. Portions of muscles (heart, tongue, leg) were fixed in 10% buffered neutral formalin and processed for histology. Paraffin-embedded sections were cut at 5 µm and examined after staining with haematoxylin and eosin (H and E).

For immunohistochemical (IHC) staining, paraffin sections were reacted with antibodies to S. speeri bradyzoites as described by Dubey and Lindsay (Reference Dubey and Lindsay1999). For transmission electron microscopy (TEM), skeletal muscle from a KO mouse euthanized 220 day p.i. (Table 1) from a formalin-fixed specimen, embedded in paraffin, was processed for TEM as described (Dubey et al. Reference Dubey, Calero-Bernal, Rosenthal, Speer and Fayer2015). Briefly, for TEM, ultra-thin sections (60–90 nm) were stained with uranyl acetate and lead citrate then examined in a JEOL JEM 1400 electron microscope.

The cryo-preserved culture of merozoites/schizonts derived from the liver of a nude mouse # 4309 infected with S. speeri from the third opossum (# 8157); collected from Argentina were used for molecular characterization. This culture was free of S. neurona and S. falcatula as evidenced by lack of infectivity to budgerigars and KO mice (Dubey et al. Reference Dubey, Speer and Lindsay2000d ).

Culture-derived merozoites/schizonts of S. speeri isolate from opossums # 8157 were sequenced at three nuclear DNA regions; 18S rRNA, 28S rRNA, and ITS1, and two mitochondrial DNA regions; cox1 and cytb. DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. DNA quantification and quality were determined by Thermo Scientific NanoDrop Lite Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

The complete regions of 18S rRNA and 28S rRNA were amplified using overlapping fragments and primer pairs; ERIB1/S2r, S5f/S4r, S3f/Primer Bsarc and KL1/LS2R, LS1F/KL3, respectively as described previously (Gjerde and Josefsen, Reference Gjerde and Josefsen2015). In addition, the complete ITS1 region and the partial sequence of cox1 and cytb loci were also amplified using primer pairs SU1F/5·8SR2, SF1/SR5 and CybF1/CybR1, respectively (Gjerde and Josefsen, Reference Gjerde and Josefsen2015). The polymerase chain reaction (PCR) amplifications were performed in 50 µL total reaction volume containing 10 pmol of each primer and 1× Taq PCR Master Mix Kit (Qiagen, Inc., Valencia). The thermal cycler (Veriti® Thermal Cycler, Applied Biosystems, Foster City, CA, USA) conditions were set at initial denaturation at 95 °C for 10 min; 40 cycles of amplification (95 °C for 45 s, 52–56 °C for 45 s and 72 °C for 1 min) and final extension at 72 °C for 10 min. Both, the positive (DNA of Sarcocystis felis) and the negative (H2O) controls were included in all the batches, respectively. The amplified PCR products were run on 2·5% (w/v) agarose gel with ethidium bromide stain and visualized using Gel Logic 212 Imaging Systems (Eastman Kodak Company, Rochester, NY, USA).

The single PCR amplicons of 18S rRNA, 28S rRNA, ITS1, cox1 and cytb were excised from the gel and purified using QIAquick Gel Extraction (Qiagen, Inc., Valencia) according to the manufacturer's recommendation. The purified PCR products were sent to Macrogen Corporation (Rockville, MD, USA) for direct sequencing using the same primer pair used in PCR amplification to obtain both reads. The resulting sequences were imported, read, edited manually if necessary, and analysed using the software Geneious version 8.0·4 (Biomatters Ltd. Auckland, NZ). The sequences obtained were aligned against each other and various Sarcocystis spp. sequences published in NCBI database to detect interspecies variation on these DNA regions, respectively.

Phylogenetic trees were estimated by the Neighbour-Joining algorithm applied to Tamura-Nei genetic distances, as implemented by Geneious version 8.0·4. A phylogenetic tree based on 18S rRNA sequences was constructed using sequences of the S. speeri isolate (opossum # 8157) and previously published sequences of various Sarcocystis spp. using software Geneious version 8.0·4. Input sequences were the 18S rRNA regions of various Sarcocystis species and related taxon retrieved from GenBank. The second phylogenetic tree was constructed based on ITS1 sequence obtained from S. speeri isolate (opossum # 8157). Input sequences were the ITS1 regions of different Sarcocystis species and related taxon retrieved from NCBI GenBank. Trees were tested by selecting bootstrap method with the value of 1000 replicates.

RESULTS

Individual metrocytes were first seen in skeletal muscle of the mouse killed on 12 days p.i. (Fig. 1A). The presence of a prominent parasitophorous vacuole helped in the recognition of these early metrocytes (Fig. 1A and B). In H and E stained sections metrocytes were stained faintly, but stained strongly with anti-bradyzoite S. speeri rabbit antibodies (Fig. 1A, C and F). In a Giemsa-stained smear of infected mouse killed day 25 p.i., sarcocysts were serpentined, up to 4 mm long and up to 40 µm wide; metrocytes were often rectangular to ovoid and 10 µm long (Fig. 1D and E). Spike-like villar protrusions on the sarcocyst wall began to appear on day 34 p.i. and were clearly visible on day 39 p.i. (Fig. 1G). Few bradyzoites had formed by 46 days p.i., but sarcocysts at day 50 p.i. contained predominantly metrocytes.

Fig. 1. Sarcocystis speeri sarcocysts in skeletal muscles of KO mice fed sporocysts. D –smear stained with Giemsa, the rest are tissue sections. A, C and F –IHC staining with anti–S. speeri antibodies, B and E stained with haematoxylin and eosin, G stained with Toluidine Blue, (A) Myocyte containing 1 metrocyte inside a vacuole (arrow), day 14 p.i. (B) A myocyte with two metrocytes (arrows), each in a separate vacuole. day 14 p.i. (C) Three immature sarcocysts. Day 17 p.i. (D, E) Immature sacrocysts with metrocytes (arrows). Day 25 p.i. The metrocytes in smear are much bigger in size than metrocytes in sections. (F) Numerous sarcocysts. Day 52 p.i. (G) Mature sarcocyst. Note villar protrusions (arrowheads) on sarcocyst wall, few metrocytes (me) and longitudinally cut bradyzoites (br). Day 222 p.i. Bar applies to all parts.

Each of two KO mice fed a 10−4 dilution of the sporocyst inoculums from opossum # 9068 was infective to KO mice; numerous bradyzoites were recovered in pepsin digests of muscles of mice 83 days p.i. Bradyzoites were not found in muscle digests of two KO mice killed 83 days after oral inoculation with 10−6 dilution of sporocysts; 10−5 dilution was not tested. Thus, at least 1000 infective sporocysts were in the inocula used to study development of sarcocysts.

Sarcocysts from KO mice killed 104, 140 and 220 days p.i. were examined ultra-structurally. The sarcocyst wall at 220 days p.i. (Fig. 2) was identical to sarcocysts illustrated previously from KO mice killed 50 – 85 days p.i. (Table 1). The sarcocyst wall from the tip of the villar protrusions to the base of bradyzoites was up to 2·5 µm thick, depending on the length of villar protrusions, the ground substance (gs) was approximately 1 µm thick (Fig. 2A). The villar protrusions were steeple-shaped surmounted by a spire, type 38 (Dubey et al. Reference Dubey, Calero-Bernal, Rosenthal, Speer and Fayer2015). The longitudinally cut bradyzoites (n = 7) in TEM sections measured 7·5–9·5 × 2·0–3·2 µm in size (Fig. 3). They contained a conoid, rhoptries, micronemes, amylopectin granules and a nucleus. Micronemes were numerous, often arranged haphazardly, and located in the 1–2 µm conoidal part of the bradyzoite; they were approximately 0·4 µm long and slender. Rhoptries were few; no more than two were identified with certainty because their contents appeared similar to those of dense granules. There was one convoluted mitochondrion. Amylopectin granules were numerous and distributed in most of the bradyzoite length, except the region containing micronemes (Fig. 3). The nucleus was up to 3 µm long and located terminally/sub-terminally. The non-conoidal end was round to conical in shape.

Fig. 2. TEM of the sarcocyst wall of Sarcocystis speeri in KO mouse, 222 days p.i. (A) Note steeple-shaped surmounted by a spire villar protrusions (vp, arrows), a thick ground substance (gs) without granules, and bradyzoites (br). (B) Higher magnification of villar protrusions marked with arrows in Fig. 2A. Note spire (sp) and absence of microtubules in vp. Abbreviation: TEM, transmission electron microscopic.

Fig. 3. TEM of a longitudinally cut bradyzoite from the sarcocyst in Fig. 2A. Note conoid (co), micronemes (mn), amylopectin granules (am), 2 rhoptries (rh), a mitochondrion (mc), dense granules (dg) and a nucleus (nu) with a prominent nucleolus (no). Abbreviation: TEM, transmission electron microscopic.

The 18S rRNA (in three fragments), 28S rRNA (in two fragments), ITS1, cox1 and cytb loci were amplified by PCR using DNA of S. speeri isolate from opossum # 8157. DNA sequencing of PCR amplicons resulted in  unambiguous sequences of three nuclear DNA regions; 18S rRNA (1751 bp), 28S rRNA (1522 bp) and ITS1 (1190 bp), and two mitochondrial DNA loci; cox1 (1057 bp) and cytb (741 bp). These sequences were submitted to GenBank with accession numbers; KT207459 (18S rRNA), KT207460 (28S rRNA), KT207458 (ITS1), KT207461 (cox1) and KT207462 (cytb).

Phylogenetic analysis based on both the 18S rRNA and the ITS1 sequences obtained from S. speeri isolate from opossum # 8157 confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona (Figs 4 and 5). The 28S rRNA, cox1 and cytb sequences of S. speeri shared the highest identity with sequences of S. neurona (AF092927) 99·0%, Sarcocystis lutrae (KM657808) 99·0% and Toxoplasma gondii strain RH (JX473253) 100%, respectively.

Fig. 4. Phylogenetic tree based on 18S rRNA sequences. Input sequences were the 18S rRNA regions of various species retrieved from GenBank, and 1751 bp long sequence obtained from S. speeri isolate. Accession number of gene sequences was given in parenthesis following the species name. Tree was built by selecting the Tamura-Nei genetic distance model and Neighbour-Joining tree methods (Geneious version 8.0·4). Tree was tested by selecting bootstrap method with value of 1000 replicates. Sarcocystis speeri was inferred to share an especially close relationship to S. neurona, another species employing opossums as its definitive host and S. ramphastosi an avian species of Sarcocystis with unknown definite host.

Fig. 5. Phylogenetic tree based on ITS1 sequences. Input sequences were the ITS1 regions of various species retrieved from GenBank, and 1190 bp long sequences obtained from Sarcocystis speeri isolate. Accession number of gene sequences was given in parenthesis following the species name. Tree was built by selecting the Tamura–Nei genetic distance model and Neighbour-Joining tree methods (Geneious version 8.0·4). Tree was tested by selecting bootstrap method with value of 1000 replicates. Sarcocystis speeri clustered together with Sarcocystis species that employs opossums as a definitive host.

A nearly complete locus of 18S rRNA sequences (1751 bp) was obtained from S. speeri isolate after PCR amplification and sequencing of three overlapping fragments. The 18S rRNA sequences obtained from S. speeri isolate shared the highest identity with sequences of S. neurona strain SN5 (U07812). Sarcocystis speeri and S. neurona (U07812) differed at 5 of 1751 aligned sites.

The ITS1 sequence obtained from S. speeri isolate also shared the highest identity with sequences of S. neurona (AF252407). Ten of 1190 nucleotide positions were recorded having variations between these two sequences (99·4% identity).Four of 10 SNPs are confirmed between S. speeri (KT207458) and S. neurona (AF252407) ITS1 sequences. The other six nucleotides differences between these two sequences indicating uncertainty or a potential of two different nucleotides at particular sites because S. speeri sequence chromatograms in both, the forward and the reverse reads recorded double peaks at these positions.

The 28S rRNA sequences of S. speeri and S. neurona (AF092927) shared the highest identity (99·0%). Sarcocystis speeri sequences chromatograms in both, the forward and the reverse reads recorded double peaks (C and G) at position 482; the C base was predominant and selected base call. 28S rRNA of S. speeri also shared the higher sequence identity with other species; S. calchasi (FJ232949), S. arctica (KF601312), Sarcocystis (Frenkelia) glareoli (AF044251), S. lutrae (KM657772) and S. rileyi (KJ396585).

The partial cox1 sequence (1057 bp) was obtained from S. speeri isolate after PCR amplification and sequencing. The cox1 sequences obtained from S. speeri isolate shared 99% identity with S. lutrae (KM657808), 98% identity with S. arctica (KF601321), 97% identity with S. rileyi (KJ396582) and 83% identity with S. hardangeri (KC209630).

The partial cytb sequences (741 bp) obtained from S. speeri isolate shared 100% identity with sequences of T. gondii strain RH (JX473253), corresponding to nucleotide positions 13–754. It did not match with any available Sarcocystis spp. cytb sequences. Identical results have been reported in the previous studies that applied same primers; CybF1/CybR1 for amplification of S. lutrae (Gjerde and Josefsen, Reference Gjerde and Josefsen2015). It was possible that the target region of these primers was more conserved portion of the gene. Limited sequences data of cytb gene from Sarcocystis spp. are available to compare.

DISCUSSION

The sarcocyst seen in the present study were structurally similar to those observed previously as described in Table 1. In the present study, additional details on development of the cyst wall and structures of bradyzoites are provided. Sarcocysts were first seen at 12 days p.i., and they became macroscopic by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50 – 220 days p.i.

To enhance the genetic characterization of this specimen, we sequenced three nuclear (18S rRNA, 28S rRNA and ITS1), and two mitochondrial loci; cox1 and cytb of S. speeri isolate were sequenced and compared with homologues in other species of Sarcocystis. Phylogenetic inferences based on sequences of 18S and ITS1 portions of rRNA confirmed a close relationship of S. speeri to other species of Sarcocystis, particularly those which also employ the opossums as their definitive hosts, i.e. S. neurona. In the phylogenetic tree based on 18S rRNA sequences, S. speeri grouped closely with S. neurona and S. ramphastosi. Sarcocystis ramphastosi is an avian species of Sarcocystis with unknown definite host (Dubey et al. Reference Dubey, Lane and van Wilpe2004). In the phylogenetic tree based on ITS1 sequences; S. speeri clustered in a clade together with S. neurona, S. falcatula, S. dasypi and S. lindsayi; all four employs opossums as the definitive host.

The 28S rRNA sequences of S. speeri shared the highest identity with sequences of S. neurona (AF092927). The cox1 sequences of S. speeri isolate shared the highest identity with sequences of S. lutrae (KM657808). These two sequences can be differentiated by 10 SNPs. The use of cox1 gene as a genetic marker for Sarcocystis species discrimination has been proposed recently, so only limited few sequences are now available for comparative use (Gjerde, Reference Gjerde2013).

These data sufficed to conclude, however, that S. speeri is very closely related other species of Sarcocystis that employ opossums as their definitive hosts. There were few SNPs that differentiated this isolate of S. speeri exemplars of S. neurona. Sarcocystis speeri, complementing certain phenotypic characteristics (i.e. development of sarcocysts in KO mice within 12 days that become macroscopic within 25 days bearing a ‘type 38’ wall structure that does not change with age) that set them apart from other known species such as S. falcatula and S. neurona.

ACKNOWLEDGEMENTS

The authors thank Mr Efrain Pérez and Joseph Madary, Joint Pathology Center, Veterinary Services, U.S. Army, Silver Spring, Maryland for excellent technical help with electron microscopy.

FINANCIAL SUPPORT

This research received no specific grant from any funding agency, commercial or not-for-profit sectors.

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Figure 0

Table 1. Transmission electron microscopic examination of sarcocysts of Sarcocystis speeri

Figure 1

Fig. 1. Sarcocystis speeri sarcocysts in skeletal muscles of KO mice fed sporocysts. D –smear stained with Giemsa, the rest are tissue sections. A, C and F –IHC staining with anti–S. speeri antibodies, B and E stained with haematoxylin and eosin, G stained with Toluidine Blue, (A) Myocyte containing 1 metrocyte inside a vacuole (arrow), day 14 p.i. (B) A myocyte with two metrocytes (arrows), each in a separate vacuole. day 14 p.i. (C) Three immature sarcocysts. Day 17 p.i. (D, E) Immature sacrocysts with metrocytes (arrows). Day 25 p.i. The metrocytes in smear are much bigger in size than metrocytes in sections. (F) Numerous sarcocysts. Day 52 p.i. (G) Mature sarcocyst. Note villar protrusions (arrowheads) on sarcocyst wall, few metrocytes (me) and longitudinally cut bradyzoites (br). Day 222 p.i. Bar applies to all parts.

Figure 2

Fig. 2. TEM of the sarcocyst wall of Sarcocystis speeri in KO mouse, 222 days p.i. (A) Note steeple-shaped surmounted by a spire villar protrusions (vp, arrows), a thick ground substance (gs) without granules, and bradyzoites (br). (B) Higher magnification of villar protrusions marked with arrows in Fig. 2A. Note spire (sp) and absence of microtubules in vp. Abbreviation: TEM, transmission electron microscopic.

Figure 3

Fig. 3. TEM of a longitudinally cut bradyzoite from the sarcocyst in Fig. 2A. Note conoid (co), micronemes (mn), amylopectin granules (am), 2 rhoptries (rh), a mitochondrion (mc), dense granules (dg) and a nucleus (nu) with a prominent nucleolus (no). Abbreviation: TEM, transmission electron microscopic.

Figure 4

Fig. 4. Phylogenetic tree based on 18S rRNA sequences. Input sequences were the 18S rRNA regions of various species retrieved from GenBank, and 1751 bp long sequence obtained from S. speeri isolate. Accession number of gene sequences was given in parenthesis following the species name. Tree was built by selecting the Tamura-Nei genetic distance model and Neighbour-Joining tree methods (Geneious version 8.0·4). Tree was tested by selecting bootstrap method with value of 1000 replicates. Sarcocystis speeri was inferred to share an especially close relationship to S. neurona, another species employing opossums as its definitive host and S. ramphastosi an avian species of Sarcocystis with unknown definite host.

Figure 5

Fig. 5. Phylogenetic tree based on ITS1 sequences. Input sequences were the ITS1 regions of various species retrieved from GenBank, and 1190 bp long sequences obtained from Sarcocystis speeri isolate. Accession number of gene sequences was given in parenthesis following the species name. Tree was built by selecting the Tamura–Nei genetic distance model and Neighbour-Joining tree methods (Geneious version 8.0·4). Tree was tested by selecting bootstrap method with value of 1000 replicates. Sarcocystis speeri clustered together with Sarcocystis species that employs opossums as a definitive host.