Microsporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups

The spore polar tube is a unique organelle required for cell invasion by fungi-related microsporidian parasites. Two major polar tube proteins (PTP1 and PTP2) are encoded by two tandemly arranged genes in Encephalitozoon species. A look at Antonospora (Nosema) locustae contigs (http://jbpc.mbl.edu/Nosema/Contigs/) revealed significant conservation in the order and orientation of various genes, despite high sequence divergence features, when comparing with Encephalitozoon cuniculi complete genome. This syntenic relationship between distantly related Encephalitozoon and Antonospora genera has been successfully exploited to identify ptp1 and ptp2 genes in two insect-infecting species assigned to the Antonospora clade (A. locustae and Paranosema grylli). Targeting of respective proteins to the polar tube was demonstrated through immunolocalization experiments with antibodies raised against recombinant proteins. Both PTPs were extracted from spores with 100mM dithiothreitol. Evidence for PTP1 mannosylation was obtained in studied species, supporting a key role of PTP1 in interactions with host cell surface.     

The microsporidian spore invasion tube. The ultrastructure, isolation, and characterization of the protein comprising the tube

The extrusion apparatus of the microsporidian parasitic protozoan Nosema michaelis discharges an invasion (or polar) tube with a velocity suitalbe for piercing cells and injecting infective sporoplasm. The tube is composed of a polar tube protein (PTP) which consists of a single, low molecular weight polypeptide slightly smaller than chymotrypsinogen-A. Assembled PTP tubes resist dissociation in sodium dodecyl sulfate and brief exposures in media at extreme ends of the pH range; however, the tubes are reduced by mercaptoethanol and dithiothreitol. When acidified, mercaptoethanol-reduced PTP self-assembles into plastic, two-dimensional monolayers. Dithiothreitol-reduced PTP will not reassemble when acidified. Evidence is presented which indicates that PTP is assembled as a tube within the spore; that the ejected tube has plasticity during sporoplasm passage; and, finally, that the subunits within the tube polymer are bound together, in part, by interprotein disulfide linkages.     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. The microsporidian invasion process involves the extrusion of a unique polar tube into host cells. Both the spore wall and the polar tube play an important role in microsporidian pathogenesis. So far, five spore wall proteins (SWP1, SWP2, Enp1, Enp2, and EcCDA) from Encephalitozoon intestinalis and Encephalitozoon cuniculi and five spore wall proteins (SWP32, SWP30, SWP26, SWP25, and NbSWP5) from the silkworm pathogen Nosema bombycis have been identified. Here we report the identification and characterization of a spore wall protein (SWP5) with a molecular mass of 20.3 kDa in N. bombycis. This protein has low sequence similarity to other eukaryotic proteins. Immunolocalization analysis showed SWP5 localized to the exospore and the region of the polar tube in mature spores. Immunoprecipitation, mass spectrometry, and immunofluorescence analyses revealed that SWP5 interacts with the polar tube proteins PTP2 and PTP3. Anti-SWP5 serum pretreatment of mature spores significantly decreased their polar tube extrusion rate. Taken together, our results show that SWP5 is a spore wall protein localized to the spore wall and that it interacts with the polar tube, may play an important role in supporting the structural integrity of the spore wall, and potentially modulates the course of infection of N. bombycis.     

Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

Ultrastructural study on a novel microsporidian, Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda)

A microsporidian pathogen, infecting the epithelial cells of the hepatopancreas of Chinese mitten crab, Eriocheir sinensis, was studied by electron microscopy. The detailed ultrastructure of life cycle of the pathogen including proliferative and sporogonic developmental stages are provided. All stages of the parasite are haplokaryotic and develop in a vacuole bounded by a single membrane in contact with host cell cytoplasm. Sporogenesis is synchronous with the same developmental stage in one vacuole. Sporogony shows a characteristic of multinucleate sporogonial plasmodia divided by rosette-like division, producing 4 or 8 sporoblasts. The mature spore is ellipsoidal, length (mean) 1.7 microm, width 1.0 microm, with a uninucleate in the center of the sporoplasm, 7 turns of the polar filament, a bell-like polaroplast of compact membranes and obliquely positioned posterior vacuole. The morphological characteristics of this novel microsporidian pathogen have led us to assign the parasite to a new species of Endoreticulatus, E. eriocheir sp. nov., that has not been reported previously from crab.     

A proteomic-based approach for the characterization of some major structural proteins involved in host-parasite relationships from the silkworm parasite Nosema bombycis (Microsporidia)

Nosema bombycis is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about host-parasite interactions at the molecular level for this spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. Major microsporidian structural proteins from the spore wall (SW) and the polar tube (PT) are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by SDS-PAGE and 2-DE to establish complementary proteomic profiles. Three proteins were shown to be located at the parasite SW. Moreover, 17 polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots were shown to correspond to polar tube proteins (PTPs) by immunofluorescent assay and transmission electron microscopy immunocytochemistry on cryosections. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by MASCOT and SEQUEST searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a nongenomic context.     

On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients. Spore invasion of host cells involves extrusion of a polar tube. After immunocytochemical identification of several polar tube proteins (PTPs) in E. cuniculi , a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced. The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques. The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats. An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP. No similarity with known proteins has been found. The protein was expressed in Escherichia coli , purified and injected into mice. The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry. Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies.     

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia)

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed‐sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5–6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.     

Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.     

A theory for the mechanism of polar filament extrusion in the microspora

A theory is presented which can explain the interaction of the major factors known to influence in vitro extrusion of the microsporidian polar filament. It is proposed that the pH, and concentration and species of cation in the external medium influence the activity of car?ylic ionophore molecules in spore membranes in the following manner: (1) Alkaline environmental conditions establish a proton gradient across the spore plasma membrane, and facilitate the activation of ionophore molecules in this membrane. (2) This proton gradient drives an ionophorically-mediated cation/proton exchange across the plasma membrane. (3) As protons are lost from the sporoplasm its alkalinity increases, so that ionophore molecules in organelle membranes (i.e. in the polaroplast and posterior vacuole) are activated. This initiates a cation/proton exchange between sporoplasm and organelles. (4) Continued movement of cations into organelles in the spore causes major osmotic imbalance across spore membranes. This leads to a rapid inflow of water into the spore and swelling of the polaroplast and posterior vacuole. The associated pressure increase in the spore causes the explosive discharge of the polar filament through the polar cap. This model is used to explain previously published results from the literature, and methods of testing predictions generated by this hypothesis are outlined.     

Brachiola algerae spore membrane systems,their activity during extrusion,and a new structural entity,the multilayered interlaced network,associated with the polar tube and the sporoplasm

The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the "thickened" plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1-h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane invaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin "unit limiting membrane", present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross-connections forming the "thick blistered" surface by 30 min-postextrusion.     

Cryptic microsporidian parasites differentially affect invasive and native Artemia spp.

We investigated the host specificity of two cryptic microsporidian species (Anostracospora rigaudi and Enterocytospora artemiae) infecting invasive (Artemia franciscana) and native (Artemia parthenogenetica) hosts in sympatry. Anostracospora rigaudi was on average four times more prevalent in the native host, whereas E. artemiae was three times more prevalent in the invasive host. Infection with An. rigaudi strongly reduced female reproduction in both host species, whereas infection with E. artemiae had weaker effects on female reproduction. We contrasted microsporidian prevalence in native A. franciscana populations (New World) and in both invaded and non-invaded Artemia populations (Old World). At a community level, microsporidian prevalence was twice as high in native compared with invasive hosts, due to the contrasting host-specificity of An. rigaudi and E. artemiae. At a higher biogeographical level, microsporidian prevalence in A. franciscana did not differ between the invaded populations and the native populations used for the introduction. Although E. artemiae was the only species found both in New and Old World populations, no evidence of its co-introduction with the invasive host was found in our experimental and phylogeographic tests. These results suggest that the success of A. franciscana invasion is probably due to a lower susceptibility to virulent microsporidian parasites rather than to decreased microsporidian prevalence compared with A. parthenogenetica or to lower microsporidian virulence in introduced areas.     

F-actin and beta-tubulin localization in the myxospore stinging apparatus of Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa, Myxosporea)

To understand the discharge mechanism of Myxozoan polar capsule (cnida) it is necessary to verify the role of major cytoskeletal proteins in the process. With this aim F-actin and beta-tubulin localization in spores of myxosporean developmental phase (in myxospores) of Myxobolus pseudodispar Gorbunova, 1936 has been studied under confocal scanning laser microscope using phalloidin fluorescent staining of F-actin and indirect anti-beta-tubulin immunostaining. F-actin has been detected in walls of the stinging tube invaginated into the polar capsule of myxospore. The fact suggests the contractile proteins involvement in the process of myxozoan polar capsule extrusion. In addition, the cytoplasm of amoeboid sporoplasm inside the spore cavity is stained by phalloidin. A polar cap with strong beta-tubulin immunoreacton is observed at the front pole of fully mature myxospore above the outlets of the polar capsule discharge channels. The role of the beta-tubulin cap is supposed to be similar to that of the cnidarian cnidocil made of microtubules. The weaker beta-tubulin immunoreactivity has been found in stinging tubes, in polar capsule walls as well as in the suture line of spore walls and in the cytoplasm of amoeboid sporoplasm. The involvement of cytoskeletal proteins in the process of polar capsule extrusion is discussed. A hypothesis on the myxozoan polar capsule discharge mechanism is suggested. The mechanism of myxozoan cnida discharge is compared with that of cnidaria.     

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.     

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.     

Unique physiology of host–parasite interactions in microsporidia infections

Microsporidia are intracellular parasites of all major animal lineages and have a described diversity of over 1200 species and an actual diversity that is estimated to be much higher. They are important pathogens of mammals, and are now one of the most common infections among immunocompromised humans. Although related to fungi, microsporidia are atypical in genomic biology, cell structure and infection mechanism. Host cell infection involves the rapid expulsion of a polar tube from a dormant spore to pierce the host cell membrane and allow the direct transfer of the spore contents into the host cell cytoplasm. This intimate relationship between parasite and host is unique. It allows the microsporidia to be highly exploitative of the host cell environment and cause such diverse effects as the induction of hypertrophied cells to harbour prolific spore development, host sex ratio distortion and host cell organelle and microtubule reorganization. Genome sequencing has revealed that microsporidia have achieved this high level of parasite sophistication with radically reduced proteomes and with many typical eukaryotic pathways pared-down to what appear to be minimal functional units. These traits make microsporidia intriguing model systems for understanding the extremes of reductive parasite evolution and host cell manipulation.     

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.