Use of random amplified polymorphic DNA-polymerase chain reaction for the definition of genetic markers for species and strains of porcine Oesophagostomum

Nodular worms are common parasites of pigs, and research has recently started to focus on the biology of these nematodes. However, the methods for delineation of species at immature developmental stages and␣for␣differentiation of various lines of the same species␣remain limited. For differentiation of porcine Oesophagostomum species and strains by genomic fingerprinting, random amplified polymorphic DNA-polymerase chain reaction was performed on DNA derived from 20 larval batches of anthelmintic-susceptible and resistant strains and isolates of these nematodes and 2 ruminant Oesophagostomum spp. Polymorphic DNA markers could be amplified with 9 of the 33 primers tested. In all, 13 markers were species-specific and 6 markers could differentiate between strains or groups of strains. With a combination of the latter, artificially selected anthelmintic-resistant strains and the susceptible mother strain of O. dentatum could be delineated. When single adult worms were compared, considerable variations between strains of the same species and between individuals from the same strain could be detected. The differentiation of Oesophagostomum strains and species at all parasitic stages on the basis of genetic markers could greatly facilitate studies on the biology of these parasites. Received: 6 January 1997 / Accepted: 24 March 1997     

Trypanosoma rangeli : increase in virulence with inocula of different origins in the experimental infection in mice

We compared two murine models of Trypanosoma rangeli infection. The same inoculum dose and age-matched hosts were used in both cases. One group was infected with trypomastigotes obtained from passages in mice and the other, with trypomastigotes obtained from cell culture after a passage in mice. We observed that trypomastigotes obtained from the in vitro cellular infection showed increased virulence in␣experimental animals, with a 70% rate of death being noted in experimental mice instead of the lack of mortality seen when in vivo-derived parasites were used. The greatest levels of parasitemia and tissual lesions in the presence of the parasite also occurred when in vitro-derived parasites were used. Received: 10 March 1997 / Accepted: 10 April 1997     

Mixed versus pure variants of desmoplastic melanoma: a genetic and immunohistochemical appraisal

Desmoplastic melanoma is subclassified into pure and mixed variants with a higher rate of lymph node metastasis in the latter. Given that reasons for these biological differences are not currently known, we investigated these subtypes with techniques that included genetic and immunohistochemical analyses of 43 cases of desmoplastic melanoma (24 pure, 19 mixed). Direct DNA sequencing was performed on BRAFV600E, RET gene (coding region on exon 11) and KIT (exons 11, 13 and 17). Immunohistochemical stains were performed with antibodies to markers of significance with respect to biological potential of nevomelanocytic proliferations and/or desmoplastic melanoma (Ki-67, CD117, nestin, clusterin, SOX10 and CD271/p75NTR). Polymorphism at the RET coding region (RETp) was noted in 33% of pure (8/24 cases) versus 24% of mixed (4/17 cases); BRAFV600E was absent in all cases of pure (0/24 cases) versus 6% of mixed (1/17 cases); no mutations were found in any of the cases on analyses of exons 11, 13 and 17 of the c-KIT gene (P=NS for all). For immunohistochemical analyses of pure versus mixed: mean percentage of Ki-67 nuclear positivity was 5% (s.d.=5.6) versus 28% (s.d.=12.6, P<0.001); CD117 stained 26% (6/23 cases) versus 78% (14/18 cases, P<0.01); nestin stained 83% (n=19/23 cases) versus 89% (16/18 cases, P=NS); clusterin stained 4% (1/23 cases) versus 6% (1/18 cases, P=NS); SOX10 87% (20/23 cases) versus 94% (17/18 cases, P=NS) and CD271 stained 61% (14/23 cases) versus 67% (12/18 cases, P=NS). Increased CD117 staining in the mixed variant suggests that alterations in the KIT protein may be involved in tumor progression. In addition, the proliferative index of the mixed variant was higher than that of the pure variant.     

Strongyloides stercoralis: identification of antigens in natural human infections from endemic areas of the United States

Using Western-blot analysis, we identified eight immunodominant antigens (apparent molecular weights 96, 86, 75, 56, 41, 32, 28, and 26 kDa) of Strongyloides stercoralis in natural human infections. For this study, 78 individual serum samples were obtained from S.␣stercoralis-infected patients residing in endemic areas of the United States. Poly A⁺ RNA was translated in vitro in the rabbit-reticulocyte lysate system. The newly synthesized translation products were immuno-precipitated with S. stercoralis human infection sera. All eight of the identified antigens were detected in the immunoprecipitates. The potential of these antigens as targets for immunodiagnosis is also discussed. Received: 28 January 1997 / Accepted: 25 April 1997     

Evaluation of the effect of peptidyl membrane-interactive molecules on avian coccidia

This study examined the lytic effect of seven different synthetic peptidyl membrane-interactive molecules (Peptidyl-MIMs) on sporozoites of five different species of Eimeria infecting chickens and merozoites of two different species that infect chickens. All Peptidyl-MIMs (pMIMs) demonstrated antiparasitic effects at concentrations of 1–50 μM during incubation periods varying from 1 to 20 min. In addition, electron microscopy showed that ultrastructural degeneration of the pellicle of sporozoite stages of the parasites occurred within 5–10 min of exposure to 5-μM concentrations of three different pMIMs. Pore-like openings were seen in the pellicle of the sporozoites at the ultrastructural level, which indicated that the pMIMs had the same mechanism of action on the parasites as that reported from studies done on bacteria. A reduction in lesion scores was seen in chickens treated orally with 10-, 50-, or ␣␣␣␣␣75-μM concentrations of two different proteolytic stabilized (methylated) pMIMs after challenge with three different species of avian coccidia in battery-cage trials. Collectively these data indicate that pMIMs may be useful in the control of coccidiosis in poultry. Received: 9 June 1998 / Accepted: 1 October 1998     

Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae)

The mitochondrial 16S rRNA gene sequences of the following eight European Metastriata tick species were obtained by direct polymerase-chain-reaction cycle sequencing and silver-staining methods: Rhipicephalus bursa, R. pusillus, R. sanguineus, R. turanicus, Boophilus annulatus, Dermacentor marginatus, Haemaphysalis punctata, and Hyalomma lusitanicum. This mitochondrial gene seems to be a good marker for the establishment of genetic relationships among closely related tick species, but it does not seem to be useful for comparisons of distantly related taxa. The molecular data provide very strong support for the monophyly of the Rhipicephalinae, including Hyalomma spp. However, the genus Rhipicephalus may not be considered a monophyletic group; in all analyses carried out in this study, R. bursa clustered with Boophilus spp. The high percentage of similarity (98.7%) observed between R.␣sanguineus and R. turanicus sequences would suggest that these species recently diverged within the Rhipicephalus genus. Phylogenetic analyses showed a monophyletic relationship among Amblyomminae taxa. The relationships between Haemaphysalis species and the true placement of this genus within Metastriata could not be resolved. Received: 12 September 1997 / Accepted: 3 December 1997     

Clonal Relationships Among Isolates of Erythromycin-Resistant <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> of Different Geographical Origin

The clonal relationships among 261 erythromycin-resistant Streptococcus pyogenes isolates collected in 1986–1997 from nine countries in Europe and North and South America were studied by using two molecular typing methods: Vir typing and random amplified polymorphic DNA (RAPD) analysis. A total of 49 different Vir genotypes (VTs) and 33 different RAPD patterns were noted among the 261 isolates. Isolates that shared the same VT and RAPD pattern were considered to belong to the same clone. Although as many as 60 different clones were found among the isolates studied, only seven clones, comprising 157 of the 261 (60%) isolates, were found in more than one country. Five of these seven clones expressed the M phenotype known to be associated with the drug efflux mechanism, and only two clones expressed the macrolide-lincosamide-streptogramin B-resistance phenotype. The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes. Furthermore, predominance of the seven clones indicates that erythromycin-resistant Streptococcus pyogenes of the same clonal origin may be widely distributed and found in several different countries. Electronic Publication     

Generation of antibody dieversity. IV. Variation within single clones of antibody-forming cells developing in vivo

Previous experimental work demonstrated that clonal variation occurs in vitro. The present experiments were designed to test for clonal variation in vivo. B cells were transferred at limiting dilution, with antigen, into irradiated recipients. Seven days later spleens were assayed for plaque-forming cell (PFC) colonies. Control experiments showed that these PFC colonies were clones, that is, they were derived from a single B cell precursor. When the clones were analyzed for heterogeneity of the PFC population, using cross-reactivity on various mixtures of red blood cells as a method of detecting differences in antibody specificity, form 23–83 % of the clones contained variants. By adjusting the amounts of helper activity and antigen available to a developing clone, we have been able to influence this variation; high levels of help and/or antigen favour pure clones, while low levels of either produce mainly mixed clones.     

Thin-layer chromatography and histochemistry analyses of neutral lipids in Helisoma trivolvis infected with four species of larval trematodes

Thin-layer chromatography (TLC) and histochemistry studies were done on the neutral lipid content of the digestive gland gonad (DGG) complex of Helisoma trivolvis infected with four species of larval trematodes. Two of the species, Ribeiroia sp. and Zygocotyle lunata, contained rediae, and the two others, Spirorchis sp. and an armatae xiphidiocercaria, contained sporocysts. The DGG infected by each species had its own distinct neutral lipid profile as determined by TLC. All profiles differed from that of the uninfected DGG. Densitometric TLC studies showed some quantitative differences in free sterols in infected versus uninfected DGGs. Visual observations of the chromatograms showed that all four species caused a marked elevation in the triacylglycerol fraction in the DGG as compared with the uninfected controls. Oil Red O (ORO) histochemistry studies showed that levels of neutral lipids were increased in the DGGs of infected versus uninfected samples. These histochemistry studies showed a variable distribution of neutral fat, ranging from its absence in the cercariae of Z. lunata and the armatae xiphidiocercaria to ORO-positive droplets in the excretory system of Ribeiroia sp. Rediae and sporocysts contained ORO-positive material in the body wall and in the space between cercariae. Received: 8 August 1997 / Accepted: 6 November 1997     

Isolation of early meiotic recombination genes analogous to S. cerevisiae REC104 from the yeasts S. paradoxus and S. pastorianus

The REC104 gene of Saccharomyces cerevisiae is required to initiate recombination in meiosis. Mutations in REC104 eliminate meiotic recombination and lead to the production of inviable spores. To determine if analogous genes exist in other yeasts, clones that hybridized to a REC104 probe were isolated from the yeasts S.␣paradoxus and S.␣pastorianus. When transformed into a rec104 strain, the REC104 analogs from these two yeasts restored spore viability and meiotic recombination to the same level as a REC104 gene cloned from S.␣cerevisiae. Compared to S.␣cerevisiae, the S.␣paradoxus gene codes for 79% identical amino acids and has 86% nucleic-acid identity in the promoter region and 84% in the coding region. The S.␣pastorianus gene codes for 63% identical amino acids and has 59% and 71% identity in the promoter and the coding regions, respectively. Received: 19 August / 19 September 1996     

In vitro phagocytosis of Giardia duodenalis cysts by hemocytes of the Asian freshwater clam Corbicula fluminea

Hemocytes of the Asian freshwater clam Corbicula fluminea, phagocytosed in vitro infectious Giardia duodenalis cysts. After 15, 30, 60, 90, and 120 min of incubation an average of 22%, 32%, 43%, 54%, and 72% of the cysts were phagocytosed by 22%, 55%, 63%, 81%, and 86% of the hemocytes, respectively. The number of hemocytes showing phagocytosis and the mean number of cysts ingested per hemocyte increased␣significantly over time (P < 0.01); the numbers of nonphagocytosed cysts significantly decreased (P < 0.02). Extrapolation reveals that C. fluminea can retain by phagocytosis an average of 1.6 × 10⁶ G. duodenalis cysts/ml hemolymph. The phagocytic capacity of C. fluminea hemocytes indicates the applicability of this freshwater benthic bivalve for bioindication of contamination of waste waters and agricultural drainage with Giardia cysts. Received: 28 March 1997 / Accepted: 6 June 1997     

Monoclonal antibodies against diagnostic Anisakis simplex antigens

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes. Received: 13 February 1997 / Accepted: 16 March 1997     

Miscibility of an oligo(p-phenylenevinylene) with polystyrene studied by solid-state nuclear magnetic resonance

Molecular proximity of cis/trans mixtures of 2,5-dimethoxy-1,4-bis[2-(3,4,5-trimethoxyphenyl)vinyl]benzene (MPV, 1 ) and polystyrene (PS) in mixtures of different MPV/PS weight ratios up to 60/40 is shown by ¹H combined rotation and multiple pulse spectroscopy (CRAMPS) using 2D exchange experiments. The MPV/PS mixtures up to a weight ratio 60/40 are homogeneous, whereas the mixture with a weight ratio MPV/PS = 80/20 is heterogeneous. The comparison of the cross polarization/magic angle spinning (CP/MAS) ¹³C NMR spectra of pure MPV and of MPV mixed with PS shows significant linebroadening in case of intimate mixing, while the heterogeneous mixture shows some extra finestructure. The miscibility results were confirmed by proton spin-locking relaxation time measurements (T_1ρH) on the mixtures. On intimate mixing, T_1ρH is averaged out and varies linearly as a function of the composition of the mixture. CRAMPS-spectra and T_1ρH measurements show that iodine-doped MPV/PS mixtures (weight ratios 40/60 and 60/40) are heterogeneous. Furthermore, doping causes a slight proton chemical shift change and reduces the T_1ρH-values of those oligomer-polymer systems.     

Chemoattraction and penetration of Echinostoma trivolvis and E. caproni cercariae in the presence of Biomphalaria glabrata, Helisoma trivolvis, and Lymnaea elodes dialysate

A petri-dish bioassay was used to study the chemoattraction and penetration of the cercariae of Echinostoma trivolvis and E. caproni in the presence of snail dialysates from Helisoma trivolvis (Pennsylvania and Colorado strains), Biomphalaria glabrata, and Lymnaea elodes. Significant chemoattraction was seen with E. trivolvis cercariae in the presence of all snail dialysates released from nonperforated dialysis sacs with a molecular-weight exclusion of 12,000. Under the same conditions, E. caproni was significantly attracted to B.␣glabrata and H. trivolvis (CO strain) but not to L.␣elodes or H. trivolvis (PA strain). Dialysis sacs were perforated with needles to allow the release of snail substances of all molecular weights into the bioassay. Cercariae of both species were significantly attracted to all snail dialysates released from perforated sacs. Moreover, cercariae entered these sacs and penetrated the snails, and 24 h later the percentage of cysts per snail species ranged from 70% to 83% for E. trivolvis and from 73% to 93% for E. caproni. Dialysates released from intact sacs were extracted in choloroform-methanol (2:1) to obtain hydrophilic and lipophilic fractions. When these extracts were placed on agar plugs in the bioassay, the lipophilic fraction, but not the hydrophilic fraction, was mainly chemoattractive. Received: 9 July 1996 / Accepted: 9 September 1996     

Susceptibility of dyskinetoplastic Trypanosoma evansi and T. equiperdum to isometamidium chloride

Isometamidium chloride (ISM) is an effective trypanocide with curative and prophylactive activity in husbandry animals. The mode of action of ISM against pathogenic trypanosomes is not fully understood, but there is evidence in the literature that kinetoplastic topoisomerase type II is selectively inhibited by the drug. This prompted the hypothesis that dyskinetoplastic trypanosomes would express a reduced level of susceptibility to ISM. From parental Trypanosoma evansi and T. equiperdum populations we generated clones containing only dyskinetoplastic organisms and clones containing organisms with kinetoplasts. The susceptibility of these clones to ISM was quantified by in vitro assays. The susceptibility of all clones was in the same range. Minor differences in drug susceptibility found between the clones showed that the dyskinetoplastic T.␣evansi and T. equiperdum clones were even more susceptible to ISM than their kinetoplastic counterparts. Thus, the hypothesis that the dyskinetoplastic trypanosomes would be less susceptible to or tolerant of ISM was clearly disproved. The previously demonstrated inhibition of kinetoplastic topoisomerase type II by ISM cannot be the primary toxic effect of the drug on trypanosomes, and the mode of action of ISM needs to be reassessed. Received: 2 April 1997 / Accepted: 14 May 1997     

The “graft versus leukemia” effect under conditions of mixed chimeric hemopoiesis

The “graft versus leukemia” effect can be induced in (C57B1/6×DBA) F₁ mice by two transplantations of cell suspensions containing bone marrow cells and splenocytes of (CBA×C57B1/6) F₁ mice and an injection of cyclophosphamide. The recipients of cell allotransplants were characterized by mixed chimeric hemopoiesis. These data outline a new approach to induction of the graft versus leukemia effect, requiring no complete elimination of the recipient's hemopoiesis and characterized by a low probability of lethal graft-versus-host reaction. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 562–565, May, 1997     

Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas

 Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal components in uterine carcinosarcoma designated as malignant mixed müllerian tumour (MMMT). We examined the biological properties of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken from a patient with uterine MMMT. We also evaluated c-myc and MYCN gene amplification in three RMS cell lines (including FU-MMT-3) derived from three MMMTs by Southern blot analysis. FU-MMT-3 cells were propagated continuously for 57 serial passages over a 2-year period in vitro. FU-MMT-3 was able to produce tumours demonstrating pure RMS in athymic nude mice. Cytogenetically, FU-MMT-3 showed a triploidy pattern, with complex karyotypic abnormalities including trisomy of chromosome 8. All three RMS cell lines, including FU-MMT-3, showed amplification of the c-myc gene (approximately fourfold to eightfold), while no cell lines demonstrated MYCN gene amplification. FU-MMT-3 is considered to provide a useful system for the study of the biological behaviour of RMS in MMMTs. Extra copies of chromosome 8 and c-myc gene amplification may be associated with the rhabdomyoblastic differentiation in MMMT. Received: 7 January 1997 / Accepted: 2 May 1997     

Evidence for the existence of genetically distinct strains of Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most frequently found microsporidium in human infections. In all, 3 distinct genotypes were detected in 12 stool samples from 8 patients with acquired immunodeficiency syndrome (AIDS). A total of 9 polymorphic sites were found in the 243-bp-long internal transcribed spacer (ITS) of the rDNA gene, whereas none was found in 241 bp of adjacent rRNA coding regions. The genotype was stable in samples taken during 11 weeks of infection from one of the patients. The existence of and the ability to discriminate among strains of E. bieneusi are important prerequisites for elucidation of the hitherto unknown reservoirs of this pathogen and the mode of its transmission and may explain its pathogenicity. Received: 18 March 1997 / Accepted: 10 April 1997     

Major histocompatibility complex class I- and II-deficient knock-out mice are resistant to primary but susceptible to secondary Eimeria papillata infections

Two distinct mechanisms seem to function in reducing oocyst output during Eimeria papillata infections in mice. For naive mice, immunity was afforded␣by␣a T-cell-independent gamma-interferon (IFN-γ) response mediated by natural killer (NK) cells. On reinfection, resistance was associated with T-cells and, to a lesser extent, perforin. To determine if antigen presentation with major histocompatibility complex␣(MHC) molecules was required to control oocyst production by NK cells during primary infection or by T-cells during secondary infection, mutant mice that lacked H2-IAβb (Aβb^−/−) or β₂-microglobulin (β2m^−/−) were used. Since MHC molecules are required for the maturation of αβ T-cells, Aβb^−/− and β2m^−/− mutant mice are also deficient in functional αβ⁺CD4⁺ or αβ⁺CD8⁺ T-cells, respectively. As compared with wild-type control mice, oocyst output by mutant mice was not significantly affected during primary infection, suggesting that the ability of NK cells to control parasite replication is not dependent on the expression of MHC molecules. On reinfection, differences were observed for mutant mice as compared with controls. Aβb^−/− mice were found to be more susceptible than β2m^−/− mice, suggesting that the αβ⁺CD4⁺ T-cell subset plays a greater role in resistance to reinfection than does the αβ⁺CD8⁺ T-cell subset. The mechanism of resistance depends on the immune status of the host and requires the coordinated interaction of both αβ⁺ T-cell subsets for optimal parasite control during subsequent infections. Received: 6 August 1997 / Accepted: 24 October 1997