Schistosoma japonicum,S. mansoni,S. haematobium,S. intercalatum,and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms

 Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN₂ only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species. Received: 1 April 1996 / Accepted: 19 June 1996     

A comparison of the susceptibility to praziquantel of Schistosoma haematobium,S. japonicum,S. mansoni,S. intercalatum and S. mattheei in hamsters

Summary Groups of six- to eight-week-old hamsters each experimentally infected with Sudanese and South African strains of S. haematobium, Puerto Rican and Liberian strains of S. mansoni, S. japonicum from mainland China, a Congo strain of S. intercalatum and a Nelspruit South African strain of S. mattheei, were treated with different dosage regimens of a new schistosomicide — praziquantel.The drug is more effective against S. haematobium when administered by the intramuscular route than per os, a complete parasitological cure being obtained following a single intramuscular injection of 200 mg/kg bwt. Per os the best results were obtained using the three highest regimens of 5×100 mg/kg, 5×50 mg/kg and 3×100 mg/kg bwt. It was observed that S. haematobium female worms are more susceptible to the compound than male worms.The results show that S. japonicum in the hamster is very susceptible to the compound, a dosage of 100 mg/kg administered orally for three days resulting in a complete parasitological cure. More than a 90% reduction in adult worms was obtained at all the dosage regimens used except 1×50 mg/kg (73.2%). Female worms were again found to be more susceptible to the drug than male worms. Hatching tests performed on ova in liver tissue of control and treated animals were positive up to four weeks after treatment, thus showing that praziquantel has no ovicidal properties.Immature S. japonicum worms were found to be markedly less susceptible to treatment than the mature schistosomes (5×100 mg/kg reduced mature adults by 99.7%, but immature forms were reduced by only 54.2%).A 100% cure rate was obtained in the hamster infected with the Liberian strain of S. mansoni following treatment with praziquantel at 3×100 mg/kg given on consecutive days, and at 3×50 mg/kg administered in one day. Treatment of the hamsters infected with the Puerto Rican strain of S. mansoni also resulted in substantial reductions of adult worms. Praziquantel was also found to be highly effective against S. intercalatum and S. mattheei.It is noted that the efficacy of the compound against S. haematobium, S. japonicum and S. mattheei in the hamster is significantly greater than that of metrifonate (Bilarcil). Following treatment with praziquantel most S. haematobium and S. japonicum worms undergo a classic hepatic shift and are trapped and killed in the liver tissue.It is considered that the present study shows that this new compound exhibits a high degree of activity against the three major schistosome species S. haematobium, S. japonicum and S. mansoni, against S. intercalatum, and against the cattle schistosome S. mattheei in the hamster, with no apparent significant differences in efficacy against the different geographical strains of the parasites used in the trials.

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Zusammenfassung Gruppen von 6–8 Wochen alten Goldhamstern wurden jeweils mit einem sudanesischen und südafrikanischen Stamm von S. haematobium, mit einem puertoricanischen und liberianischen Stamm von S. mansoni, mit S. japonicum aus Kontinentalchina, S. intercalatum vom Kongo und mit S. mattheei aus Nelspruit, Südafrika, infiziert und mit verschiedenen Dosierungen des neuen Schistosomenmittels Praziquantel behandelt.Praziquantel ist gegen S. haematobium bei intramuskulärer Gabe wirksamer als bei oraler Gabe. Eine parasitologische Heilung konnte durch eine einmalige intramuskuläre Injektion von 200 mg/kg erreicht werden. Bei oraler Gabe ergaben die drei höchsten Dosen 5×100, 5×50 und 3×100 mg/kg die besten Ergebnisse. Es wurde beobachtet, daß die Weibchen von S. haematobium empfindlicher gegen Praziquantel sind als die Männchen.Die Ergebnisse zeigen, daß S. japonicum im Goldhamster sehr empfindlich gegen Praziquantel ist. Eine parasitologische Heilung wird durch 3×100 mg/kg erreicht. Mit allen verabreichten Dosen wurde die Zahl der adulten Parasiten um mehr als 90% reduziert. Nur bei Gabe von 1×50 mg/kg betrug die Parasitenreduktion 73,2%. Wieder waren die Weibchen empfindlicher gegen Praziquantel als die Männchen. Der Miracidienschlüpfversuch wurde an Eiern aus der Leber behandelter und unbehandelter Kontrolltiere durchgeführt. Er war bis zu vier Wochen nach der Behandlung positiv; Praziquantel wirkt also nicht ovizid.Jugendliche S. japonicum waren gegen die Behandlung deutlich unempfindlicher als adulte Schistosomen (5×100 mg/kg bewirkten bei Adulten eine Reduktion um 99,7%, bei den Jugendlichen eine um 54,2%).Eine parasitologische Heilung wurde bei Goldhamstern, die mit einem liberianischen Stamm von S. mansoni infiziert waren, mit 100 mg/kg (verabreicht an drei aufeinanderfolgenden Tagen) und mit 3×50 mg/kg/die erreicht. Die Behandlung von Goldhamstern, die mit einem puertoricanischen S. mansoni-Stamm infiziert waren, ergab ebenfalls eine sehr starke Reduktion der Anzahl adulter Schistosomen. Gegen S. intercalatum und S. mattheei war Praziquantel ebenfalls sehr gut wirksam.Es wurde gefunden, daß die Wirksamkeit von Praziquantel gegen S. haematobium, S. japonicum und S. mattheei im Hamster deutlich besser ist als die von Metrifonat (Bilarcil). Nach Behandlung mit Praziquantel zeigen die meisten S. haematobium- und S. japonicum-Würmer eine klassische liver shift und werden im Lebergewebe festgehalten und abgetötet.Die Untersuchung zeigt, daß Praziquantel gegen alle drei wichtigen Schistosomenarten, S. haematobium, S. japonicum und S. mansoni, und auch gegen S. intercalatum sowie gegen den Rinderparasiten S. mattheei im Hamster hoch wirksam ist. Zwischen den in dieser Untersuchung verwendeten Parasitenstämmen unterschiedlicher geographischer Herkunft bestehen in der Wirksamkeit keine wesentlichen Unterschiede.

     

A technique for identification of cercariae of Schistosoma haematobium, S. curassoni, S. bovis and S. intercalatum

The chaetotaxy of 84 samples or isolates of Schistosoma spp. from western or central Africa has been studied. Three indices were calculated for cercariae of each sample; their average value, the skewness and kurtosis of each indice was established. Each species (S. haematobium, S. curassoni, S. bovis and S. intercalatum) was discriminated with nine variables. The present work gives information to assess, specific diagnosis with simple calculations easily achieved on a small computer.     

Functional homology of virulence plasmids in Salmonella gallinarum, S. pullorum, and S. typhimurium.

The virulence-associated plasmids of strains of Salmonella gallinarum and S. pullorum were transferred separately by mobilization with the F plasmid into virulence plasmid-cured derivatives of S. gallinarum, S. pullorum, and S. typhimurium and into a prototrophic Escherichia coli K-12 strain. The transconjugants were tested for virulence in chickens of different ages and in mice. The S. gallinarum and S. pullorum plasmids were able to restore full virulence in the three Salmonella strains, thus demonstrating functional homology in virulence plasmids from these Salmonella serotypes and biotypes. The virulence phenotypes of the transconjugants remained the same as that of the parent strain of the recipient. This, together with the fact that E. coli K-12 containing either of the virulence plasmids was avirulent for chickens, suggested that in addition to plasmid genes, chromosomal genes are important in determining virulence, particularly in determining the ability to survive and multiply in the cells of the reticuloendothelial system. The virulence plasmids were not self-transmissible and could not be transduced by temperate bacteriophages lysogenizing field strains of S. gallinarum. They were not in the same incompatibility group as F but were fi+.     

Diversity of Staphylococcus Species Strains Based on Partial kat (Catalase) Gene Sequences and Design of a PCR-Restriction Fragment Length Polymorphism Assay for Identification and Differentiation of Coagulase-Positive Species (S. aureus,S. delphini,S. hyicus,S. intermedius,S. pseudintermedius,and S. schleiferi subsp. coagulans)

A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase (kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, and S. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA, rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and <5 Euros for each strain tested) allowed unambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudintermedius CPS species.Staphylococci are a group of microorganisms occurring widely in nature. As reported in the List of Prokaryotic Names with Standing in Nomenclature (www.bacterio.net) as of the full update in March 2006, the Staphylococcus genus includes 39 valid species, 11 of which are divided into two or more subspecies, resulting in more than 50 recognized systematic entities (15).The genus includes both human and animal pathogens, generally coagulase-positive staphylococci (CPS) such as S. aureus, S. intermedius, S. delphini, S. hyicus, S. schleiferi subsp. coagulans, and S. pseudintermedius (12, 21, 29, 35), and coagulase-negative staphylococci (CNS) such as S. equorum, S. xylosus, S. carnosus, S. simulans, S. saprophyticus, S. succinus, S. warneri, S. vitulinus, S. pasteuri, S. epidermidis, and S. lentus.Notwithstanding their valuable role in food fermentation (5-9, 23, 24), some CNS such as S. saprophyticus, S. epidermidis, and S. haemolyticus exhibit increasing abilities as opportunistic/emerging pathogens in colonizing animal and human tissues, due in part to the ability of these species (particularly S. epidermidis) to form protective biofilms and their ubiquitous occurrence in the environment (37). Finally, the occurrence of some pathogenic species (S. aureus, S. intermedius, and S. hyicus) in food is a potential public health hazard, since many strains can produce enterotoxins (4, 9, 31). Indeed, enterotoxin-producing food-associated CNS strains (S. carnosus, S. equorum, S. piscifermentans, and S. xylosus) were also recently described by Zell et al. (40). Despite the importance of accurate identification of staphylococcal species by microbiological laboratories, previous studies demonstrated the unreliability of phenotypic methods, including the Vitek 2 system (bioMérieux, Marcy l''Etoile, France), compared to different molecular techniques (5-8, 11) to identify staphylococci.Due to the high sensitivity and specificity they provide, molecular markers are an alternative tool for accurate identification and classification of Staphylococcus species. Evaluations of 16S to 23S rRNA gene polymorphisms by PCR and PCR-denaturing gradient gel electrophoresis (DGGE) analyses of 16S ribosomal sequences have been assessed for the ability to achieve clear identification of strains within the Staphylococcus genus (6). Molecular assays targeting some housekeeping genes such as hsp60 (17), the 16S rRNA gene (14, 18), femA (36), tuf (22), gap (38, 39), sodA (26), rpoB (13), and dnaJ (20, 30) have been used for reliably identifying and classifying staphylococci. However, except for the sodA, hsp60, and nuc gene sequence analyses carried out by Sasaki et al. (29), which allowed some phenotypically identified S. intermedius strains to be reclassified as S. delphini and S. pseudintermedius, to our knowledge, no method allowing rapid identification and differentiation of CPS species including the recently described S. delphini and S. pseudintermedius is available to date.In this study, we evaluated the catalase (kat) gene as a new target for phylogenetic analysis of staphylococci. Catalase is a heme-containing enzyme involved in dismutation of hydrogen peroxide generated during cellular metabolism to water and molecular oxygen. In S. aureus, a correlation between catalase activity and virulence has been observed, suggesting a role for catalase in defensive mechanisms against the oxygen radicals produced by macrophages (28). Additionally, a recent study (25) showed that the S. aureus catalase is a major factor in S. aureus defense against Streptococcus pneumoniae due to neutralization of secreted H₂O₂ produced by the latter microorganism. Sanz et al. (28) showed that catalase deficiency in S. aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene. Therefore, catalase-negative staphylococcal strains may also harbor the catalase gene. Barrière et al. (3) described the katA gene of S. xylosus C2a and supposed the presence of a second catalase gene (katB) in this strain. In fact, the katA-deficient mutant of S. xylosus constructed by these authors still exhibited catalase activity. After analysis of polymorphism within the kat genes of 26 different staphylococcal species, we designed and successfully applied a molecular assay allowing rapid and unequivocal identification and differentiation of CPS species and subspecies.     

Antifungal susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii complex identified in Brazil

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.     

Schistosoma haematobium,S. intercalatum,S. japonicum,S. mansoni,and S. rodhaini in mice: relationship between patterns of lung migration by schistosomula and perfusion recovery of adult worms

The development of five schistosome species was compared in mice by the recovery of schistosomula from chopped lung tissue and of adult worms by portal perfusion. Three developmental patterns appeared. (1) Schistosoma japonicum was unique in showing an early establishment of schistosomula in and a rapid departure from the lungs together with the highest worm recovery; (2) S. haematobium contrasted by establishing later and persisting in the lungs for at least 2 weeks while yielding the lowest adult worm recovery; and (3) S. intercalatum, S. mansoni, and S. rodhaini had an intermediate pattern – they resided in the lungs for several days, then disappeared and produced intermediate numbers of adults. Lung petechiae, known to accompany the migration of S. japonicum, were never detected after infection with the other species. We speculate that the three migration patterns of schistosomes are related to the size of the relative spectra of naturally infected definitive hosts. Received: 31 August 1997 / Accepted: 15 October 1997     

Virus “Tamdy” — A new arbovirus,isolated in the Uzbec S.S.R. and Turkmen S.S.R. from ticksHyalomma asiaticum asiaticum Schulce et Schlottke, 1929, andHyalomma plumbeum plumbeum Panzer, 1796

Summary Eleven virus strains were isolated from ticksHyalomma asiaticum asiaticum Schulce et Schlottke, 1929, andHyalomma plumbeum plumbeum Panzer, 1796, collected in 1971–1974 in desert regions of the Uzbec S.S.R. and the Turkmen S.S.R. According to CF test the strains were closely related to each other and not antigenically connected with viruses from antigenic groups A, B, California, CHF-Congo, Bakau, Bunyamwera, Ganjam, Kaisodi, Qalyub, Kemerovo, Quaranfil, Simbu, Turlock, Hughes, Uukuniemi, Tete and 21 ungrouped viruses isolated from ticks. The virus was named Tamdy after the place of isolation of a prototype strain LEIV-1308 Uz. The virus does not agglutinate goose erythrocytes, it is pathogenic for suckling mice and 3 weeks old mice by intracerebral infection. Replication of a virus with CPE in cell cultures—L, Rh, A₁—and without CPE—in pig embryo kidney cell cultures—was demonstrated. According to ultra-filtration and electron microscope data the size of the virus is about 90 nm.It is rather sensitive to lipid solvents and is an RNA-virus. Morphologically the virus resembles the Bunyaviridae.With 1 Figure     

Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest

Sporothrix schenckii is the species responsible for sporotrichosis, a fungal infection caused by the traumatic implantation of this dimorphic fungus. Recent molecular studies have demonstrated that this species constitutes a complex of numerous phylogenetic species. Since the delineation of such species could be of extreme importance from a clinical point of view, we have studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strains of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences. The molecular analysis revealed that Sporothrix albicans, S. inflata, and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phenetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, and S. mexicana. The key phenotypic features for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37 degrees C, and the assimilation of sucrose, raffinose, and ribitol.     

Morphology of the teguments of Schistosoma haematobium; comparison with S. curassoni, S. bovis and S. intercalatum

Study by SEM of the anterior dorsal teguments of male Schistosoma haematobium from infected rodents. Only paired males, at least hundred days post infection, display a typical morphology. Differentiation from other closely related species obtained experimentally from rodents is possible: bovis: no spines on the tubercles; haematobium: tubercles 10 to 15 microns wide with closely packed spines; curassoni: tubercles over 15 microns wide, with large, closely packed spines; intercalatum: tubercles under 10 microns wide, with scattered spines. It is suggested that the three haematobium genotypes A, B and D are slightly different: A: pointed spines, numerous small additional spines between the tubercles; B: pointed spines, no small additional spines between the tubercles; D: blunt spines. Moreover, the lengths of the prepatent periods in the molluscs of the three S. haematobium genotypes are possibly different: A 72-86 days, B 38-46 days, D 55-58 days. The differentiation of A, B and D is supported by limited data and conclusions on this particular aspect are presented only as a working hypothesis.     

Observations on some isoenzymes of strains ofSchistosoma bovis; S. mattheei,S. margrebowiei,andS. leiperi

Summary There are two main and two weak fractions of malate dehydrogenase isoenzyme common toSchistosoma bovis, S. leiperi, S. margrebowiei andS. mattheei: a major fraction at pI 8.56, seconded at pI 7.38, with weaker activity at pI 7.05 and pI 8.15. Variation in malate dehydrogenase occurs in some species/strainsen passage.In the strains ofS. bovis there are eight common bands of lactate dehydrogenase isoenzymes, at pI 6.85, 7.05, 7.16, 7.35, 7.50, 7.95, 8.03 and 8.38 with peak activity at pI 8.38 forS. bovis [Kisat, Kajulu (B. africanus) (B. forskali), Urudi-Kenya: Sardinia; Spain].S. mattheei has a peak band at pI 8.38, and also additional bands at pI 7.16, 7.50, 7.95, 8.52, 8.65 and 8.72.S. leiperi has peak activity at pI 7.74, and about 25% of the total activity is found between pI 8.38 and pI 9.50.S. margrebowiei shows peak activity at pI 8.38, seconded by a band at pI 8.86.The strains ofS. bovis show peak activity of acid phosphatase at pI 6.45;S. mattheei andS. leiperi at pI 7.52 andS. margrebowiei at pI 7.37. Despite intraspecific variation, the secondary bands place strains ofS. bovis from Western Kenya into one group and the strains from the Mediterranean area into another group, with the exception ofS. bovis Urudi, Kenya which, like the Mediterranean forms, possesses alkaline fractions above pI 7.50.S. bovis Iran does not possess fractions alkaline to pI 6.65 and therefore shows similarity to four strains from Western Kenya.