Immunoglobulin subclass responses of wild brown rats to Sarcocystis singaporensis.

Immunoglobulin subclass responses of wild brown rats (Rattus norvegicus) from southeastern Asia to the endemic cyst-forming coccidian Sarcocystis singaporensis were characterised. The antibody response of brown rats to wild-type parasites (high reproductive capacity) showed a Th1 profile during acute infection, namely elevated concentrations of parasite-specific IgG2b and IgG2c and absence of IgG1. Chronic infection (bradyzoite development) resulted in a mixed Th1/Th2 pattern whereby significant concentrations of IgG1 appeared. A primary infection with 1000 sporocysts eight days before challenge induced protection, accompanied by significant concentrations of IgA and IgG2, particularly IgG2a. Western blot analysis of rat sera, using sporozoite and bradyzoite-extracts as antigen, revealed that IgG1, IgG2a, and IgG2b predominantly recognised molecules between 70-80 kDa in one or the other stage. Some of the antibodies were possibly directed against a 79 kDa heat shock protein of sporozoites. An apparent unresponsiveness to molecules in the low molecular weight range, particularly of bradyzoite antigens, was observed. This was abrogated by infection of rats with an avirulent strain of S. singaporensis (low reproductive capacity) indicating that a parasite that was less adapted to its host provoked a stronger immune response. These results suggest the existence of an immune evasion strategy used by Sarcocystis and show that wild rodents may be suitable as immunological research objects, reflecting the natural situation.

Reduction of transmission stages concomitant with increased host immune responses to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence.

Parasite virulence (pathogenicity depending on inoculum size) and host immune reactions were examined for the apicomplexan protozoan Sarcocystis singaporensis. This parasite is endemic in southeastern Asia and multiplies as a proliferation (merozoite) and transmission stage (bradyzoite) in rats. Virulence in wild brown rats of parasites freshly isolated in the wild (wild-type) was surprisingly constant within the endemic area and showed an intermediate level. In contrast, serially passaged parasites either became avirulent or virulence increased markedly (hypervirulence). Production of transmission stages was maximal for the wild-type whereas numbers were significantly reduced for hypervirulent and avirulent (shown in a previous study) parasites. Analyses of B and T cell immunity revealed that immune responses of WKY rats to the transmission stage were significantly higher for hypervirulent than for wild-type parasites. These results suggest that it is the immune system of the host that is not only responsible for reduction of transmission stages in individual rats, but also could act as a selective force that maintains intermediate virulence at the population level because reduction of muscle stages challenges transmission of S. singaporensis to the definitive host. Collectively, the presented data support evolutionary theory, which predicts intermediate rates of parasite growth in nature and an 'arms race' between host immunity and parasite proliferation.

Biological control of rodents using Sarcocystis singaporensis.

Parasites have been identified as an important factor in regulating vertebrate populations. In replicated field experiments (plots up to 4 ha) performed in Thailand we tested whether commensal and field rodents could be artificially infected and controlled with the host-restricted apicomplexan protozoon Sarcocystis singaporensis which is endemic in Southeast Asia. When bait-pellets containing high numbers of these parasites were consumed by rodents of three species (Rattus norvegicus, Rattus tiomanicus, Bandicota indica) in different agricultural habitats (chicken farm, oil palm plantation, ricefield), we observed a parasite-induced mortality ranging from 58% to 92%. Detection of merozoites of S. singaporensis in lung tissue samples of rats collected dead at the experimental sites using a species-specific monoclonal antibody confirmed that S. singaporensis was the causative agent of mortality. As observed with brown rats, the parasite's effect on the host was not related to sex. These experiments demonstrate for the first time that artificial infection of rodents with an endemic protozoon has the potential for effective population control.

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana.

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.

Cyclic transmission of Sarcocystis gerbilliechis n. sp. by the Arabian saw-scaled viper, Echis coloratus, to rodents of the subfamily gerbillinae.

Infection experiments with rodents and snakes were performed to elucidate the life cycle of a Sarcocystis isolate found in an Arabian saw-scaled viper, Echis coloratus. Oocytes in feces of the naturally infected and of 2 experimentally infected Arabian saw-scaled vipers were already sporulated and contained 2 sporocysts each, measuring 12.7 (12.3-13.3) microns x 11.0 (10.7-11.4) microns. After oral inoculation of various rodent species with these sporocysts, sarcocysts developed in the esophagus and skeletal muscles of gerbils and related genera. Mature sarcocysts were filiform in shape and reached a maximum length of 11.7 mm after 5 mo postinoculation (PI), whereas the width did not exceed 190 microns. The primary cyst wall formed small, knoblike protrusions, which were up to 180 nm long and 120 nm wide. Mature schizonts were present in the liver and other organs of gerbils between 11 and 14 days PI. After inoculation of vipers of 3 different genera with mature sarcocysts from gerbils, oocysts developed in the intestine of Arabian saw-scaled vipers. A comparison of these data with those from previously described Sarcocystis species with snake-rodent life cycles suggests that Sarcocystis gerbilliechis is a new species.

Sarcocystis singaporensis: studies on host specificity, pathogenicity, and potential use as a biocontrol agent of wild rats.

Host specificity and pathogenicity of Sarcocystis singaporensis were investigated as a prerequisite to a subsequent application of the parasite as a biocontrol agent of wild rats in Egypt. After inoculation of 7 snake species comprising the families Elapidae, Viperidae, Colubridae, and Boidae with sarcocysts, sporocyst development was only observed in a reticulated python. Among amphibians, reptiles, and rodents that orally received various sporocyst doses in the laboratory, 2 x 10(4) sporocysts or more were lethal to roof rats Rattus rattus frugivorous, brown rats Rattus norvegicus, and bandicoot rats Nesokia indica. Sarcocysts developed in Rattus spp. and Nile grass rats Arvicanthis niloticus. Subsequently, the pathogenicity of S. singaporensis was tested under natural control situations offering bait pellets containing high amounts of sporocysts to a free-living population of roof rats, which was monitored by indirect census baiting commonly used in rodenticide evaluation. Ten days after consumption of the bait pellets, the infected population collapsed, leading to a control success of 73%. A negative control population, which received a placebo, remained stable. These data demonstrate for the first time that S. singaporensis can be used as a biocontrol agent of wild rats. However, an immunization experiment with roof rats in the laboratory showed that these are capable of mounting a rapid specific immune response resulting in survival of acute sarcocystosis.

Sarcosporidiasis in rodents from Thailand.

One to six Sarcocystis spp. were identified in the skeletal muscles of 41 (33%) of 124 wild rodents (Rattus spp. and Bandicota indica) mainly captured in the central plains of Thailand throughout the year in 1995. Included were S. singaporensis, S. villivillosi, and S. murinotechis-like cysts all of which showed a striated cyst wall at the light microscopical level, and Sarcocystis cymruensis, S. sulawesiensis, and S. zamani which possessed smooth cyst walls. The ultrastructure of the cyst wall and other morphological characteristics used to distinguish species are described. By inoculation of muscle cysts from wild-caught rodents into coccidia-free pythons (Python reticulatus, P. molurus bivittatus), we confirmed that P. reticulatus is a suitable definitive host for S. singaporensis and S. zamani in Thailand. Furthermore, we showed by fecal examination of reticulated pythons collected in the wild and subsequent experimental infection of laboratory rats that these hosts also are naturally infected with both species. Sarcocystis cymruensis is reported for the first time from Southeast Asia. This parasite was prevalent in brown rats (Rattus norvegicus) and bandicoot rats (B. indica) which were captured near human habitations; it is likely to be transmitted to rats via cats. The definitive hosts of S. sulawesiensis and S. murinotechis are unknown. Hence, at least three Sarcocystis spp. (S. singaporensis, S. zamani, S. villivillosi) are likely to cycle between snakes and rodents in agricultural areas in Thailand. Among these, S. singaporensis appears to be the most prevalent species.

[Salivary duct carcinoma]. / Speichelgangkarzinome.

This tutorial focuses on salivary duct carcinoma (SDC), a rare, high grade neoplasm mainly of major salivary glands. The clinical course of these tumors is characterised by extended local disease, early distant metastasis, and poor outcome. The morphology of SDC is reminiscent of breast ductal carcinomas and may occasionally cause diagnostic problems. In spite of mimicry with ductal carcinoma in situ of the breast and an in situ component, that is evident in most tumors by immunohistology with antibodies directed against high molecular weight cytokeratins (Ck), SDC is always an invasive carcinoma. By immunohistology, most tumors show reactivity with antibodies directed against Ck 7, Ck 8/18 and Ck 19 whereas a morphologically indistinguishable subgroup expresses Ck 5/6 in tumor cells in addition to residual basal epithelia. Carcinoembryonic antigen, GCDFP-15 and androgen receptor are other helpful markers in routine diagnosis of SDC. Prostate-specific antigen is detectable in some cases. Abnormal p53 expression seems to indicate an adverse prognosis. Expression of c-erbB2, the over-expression of which is associated with a poor prognosis, may form the basis for a targeted therapeutic approach for selected cases of SDC.

[Myoepthelial tumors of salivary glands]. / Myoepitheliale Tumoren der Kopfspeicheldrüsen.

This tutorial focuses on myoepithelial tumors of salivary glands, an entity with heterogeneous cytomorphology and inconsistent immunophenotype. Moreover, the clinical course cannot be predicted reliably from cytomorphological and immunophenotypic analysis. This heterogeneity causes problems in routine diagnostic, so that diagnosis ultimately rests on conventional histology. In a representative series of myoepitheliomas and malignant myoepitheliomas, antibodies against cytokeratins 5/6, S 100 protein and vimentin produced the most consistent reactivity profile. Staining for cytokeratins 5/6 is a useful addition to the established immunohistologic marker panel in the work-up of myoepitheliomas, because of its reliable expression in most cases and because it may underline the epithelial nature of the lesion. Comparative genomic hybridisation (CGH) profiles of myoepitheliomas and myoepithelial carcinomas showed no chromosomal aberration in less than 50% of myoepithelial carcinomas, so that CGH is of limited help in a given case. In a case that was represented in three separately localized manifestations of the disease that differed in their CGH profiles, gross genetic aberrations suggest to be acquired during tumor progression and should raise the suspicion of malignancy. Thus, diagnosis of myoepithelial tumors of salivary glands has to rest on morphological grounds with support of a restricted panel of immunohistologic markers.

In vitro cultivation of the vascular phase of Sarcocystis singaporensis.

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (+/- 3.8) microns in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (+/- 10.6) microns and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm2 flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.

[Preliminary results for superficial parotidectomy using the ultrasonically activated scalpel (Ultracision Harmonic Scalpel)]. / Erste Ergebnisse zur Technik der laterofazialen Parotidektomie mit dem ultraschallaktivierten Skalpell (Ultracision Harmonic Scalpel(R)).

BACKGROUND: The high density of blood vessels in the parotid gland, the direct vicinity to the facial nerve and the narrow surgical situs require efficient intra- and postoperative hemostasis. The ultrasonic scalpel (US) accomplishes both tissue dissection and vessel coagulation simultaneously by an ultrasonically activated shear movement of 55 500 Hz. The combination of hemostasis and tissue dissection particularly qualifies US for use in surgery of the parotid gland. Until now, there have been no published studies about application of US in surgery of the parotid gland. METHOD: 20 patients with benign parotid tumors treated with a conventional technique of superficial parotidectomy and 20 patients with a superficial parotidectomy using the ultrasonically activated scalpel were examined in regard to intra- and postoperative hemostasis, wound healing and postoperative pain. Also, the quality of the histopathological specimen obtained by US was evaluated. RESULTS: In 85 % (17/20) of superficial parotidectomy efficient intraoperative hemostasis did not require electrocoagulation. In 15 % (3/20) of parotid tumors additional bipolar electrocoagulation were required because of venous bleeding from vessels exceeding 2.0 mm in diameter. Postoperative bleeding did not occur at all. Wound healing was uneventful in all cases. Surgery-related postoperative pain was not intensified. Time of operation was shortened. Histopathological evaluation, especially in the margin area, was not impaired. CONCLUSION: The US offers tissue dissection with effective intra- and postoperative hemostasis. The combination of simultaneous tissue dissection and hemostasis enables a good overall view and control of the surgical site. In addition, based on the mechanical function the US has only a minimal thermal effect on neighboring tissues and enables controlled tumor resection without damaging the facial nerve.

[The "Ultracision Harmonic Scalpel" ultrasound activated scalpel. Initial results in surgery of the tongue and soft palate]. / Das ultraschallaktivierte Skalpell "Ultracision Harmonic Scalpel." Erste Ergebnisse bei der Chirurgie der Zunge und des weichen Gaumens.

The ultrasonically activated scalpel (UAS) performs tissue dissection and coagulation simultaneously by an ultra-high-frequency movement of the blade. In the present prospective study, results of UAS in the surgery of the tongue and the soft palate are analysed. 25 patients with carcinoma of the tongue and 11 patients with carcinoma of the soft palate were examined in regard to intra- and postoperative hemostasis, wound healing and postoperative pain. Also, the quality of the histo-pathological specimen obtained by UAS was evaluated. In 68% (17/25) of partial tongue resections, and in 82% (9/11) of soft palate resections efficient intraoperative hemostasis did not require electrocoagulation or suture ligature. In 32% (8/25) of tongue malignancies, all with extension to the tongue base, and in 18% (2/11) of soft palate resections additional ligation was required because of arterial bleeding from vessels exceeding 1.0 mm in diameter. Wound healing was uneventful in all cases. The histopathological evaluation, especially in the margin area, was not impaired. The UAS offers a tissue dissection with efficacious intra- and postoperative hemostasis. Arterial bleeding from vessels exceeding 1.0 mm in diameter should be sutured additionally. The combination of simultaneous tissue dissection and hemostasis enables a good overall view and control of the surgical situs. In addition, based on the mechanical function the UAS has only little thermical effect on neighbouring tissues and enables a controlled tumor resection without damaging vital structures.

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae.

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.

Binding of a monoclonal antibody to sporozoites of Sarcocystis singaporensis enhances escape from the parasitophorous vacuole, which is necessary for intracellular development.

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.

Identification of a subpopulation of merozoites of Sarcocystis singaporensis that invades and partially develops inside muscle cells in vitro.

The affinity of merozoites of Sarcocystis singaporensis obtained from the lungs of acutely infected rats to muscle cells and other cell lines grown in vitro was examined. Two distinct types of mature schizonts developed in the lungs 11-13 days p.i. with sporocysts: those containing PAS- merozoites (type 1) which mainly reacted with antibodies prepared against sporozoites, and others containing PAS+ merozoites (type 2) which were antigenically close to bradyzoites. When inoculated onto cell cultures, type 1-merozoites induced schizogonic development in brain capillary endothelial cells of the rat. In contrast, type 2-merozoites invaded L6 myoblasts. In long-term cultures (50 days) of L6 cells, zoites transformed to a 8-15 microns long uninucleate stage which, tentatively, could be unizoite sarcocysts. Although the observed dichotomy in merozoite development is unprecedented in this form, evidence from previous work suggests that these observations are relevant to other Sarcocystis species. The presented cell culture system could be a first step towards successful growth of sarcocysts in vitro.