Showing papers in "Journal of Parasitology in 1999"

How much human helminthiasis is there in the world

Abstract: The impact of Norman Stoll's presidential address to the American Society of Parasitologists in Boston at Christmas time in 1946 has been and continues to be immense. Since its publication in 1947, have any helminthologists not read it? Have any parasitology text books not cited it since then? Does any parasitology course today not refer students to it? Despite our expanding knowledge of DNA, molecular biology, and immunology and the application of mathematical techniques to our understanding of epidemiology and helminth population biology, Stoll's appraisal of the wormy world still excites and challenges our imaginations. A measure of the influence of Stoll's paper is the fact that we regularly use the title of "This Wormy World" as we seek to draw attention to the global public health significance of human helminth infections. CRC Press reprinted the paper (renamed as "The Wormy World") and supplied a commentary in each of a series of volumes dealing with zoonoses (CRC, 1982). The World Health Organization used the same title for an issue of its official magazine (WHO, 1984) and recently Parasitology Today devoted an issue to revisiting the subject of "This Wormy World" in which Bundy (1997a) explained why Stoll's paper has been supremely important in directing our approach to the study and management of human helminth infections and colleagues evaluated the current status of several major helminthiases. Only last year, Bundy and de Silva (1998) again wrote about this wormy world, this time with themes of optimism and encouragement over prospects for control. In this article, I have sought to place Stoll's evaluation of human helminthiasis in a contemporary setting. I have tried to address some of the questions he asked and to review aspects of current progress in the control of human helminthiasis. Most of all, I hope this paper will be seen as a tribute to Stoll's inspirational leadership and will serve as a reminder of our need to tackle what he saw as an "unremittingly corrosive" burden of disease.

An Eimeriid origin of isosporoid coccidia with Stieda bodies as shown by phylogenetic analysis of small subunit ribosomal RNA gene sequences.

Abstract: Morphological and life cycle features of the tissue cyst-forming coccidia have been difficult to interpret in devising taxonomic classifications for the various genera. In this study, we amplified the full small subunit rRNA gene sequence of Isospora robini McQuistion and Holmes, 1988, and the partial sequence of Isospora gryphoni Olsen, Gissing, Barta, and Middleton, 1998 by PCR. Both of these species vary from Isospora species of mammals in having Stieda bodies on the sporocysts. The sequences were cloned and sequenced and were incorporated into an alignment with other Isospora species lacking Stieda bodies as well as with other coccidia. Maximum parsimony analysis of these sequences produced a single most parsimonious tree that placed I. robini and I. gryphoni in a clade containing various other eimeriid species. The Isospora species lacking Stieda bodies were in the sarcocystid clade. Similar results were found by maximum likelihood analysis. These findings indicate that the genus Isospora as defined by several authors is polyphyletic. Taxonomic changes to the genus Isospora would have to incorporate the 2 major clades found by molecular phylogenetic analysis. Isospora species with Stieda bodies should be classified in the family Eimeriidae, whereas those without Stieda bodies should remain in the family Sarcocystidae.

Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences.

Abstract: Sequences representing approximately 1,700 base pairs of the 18S rRNA gene from 10 different species in the genus Myxobolus were found to group them into 3 clusters that showed little correlation with spore morphology and size or host specificity, criteria currently used for both higher and lower taxonomic placements in the Myxozoa. Of the phenotypic criteria examined, tissue tropism was most correlated with the rRNA groupings observed. Spores of similar size and shape (Myxobolus cerebralis vs. Myxobolus squamalis) were distantly related in some instances, whereas spores with divergent morphology and size were sometimes found to be closely related (M. cerebralis and Myxobolus insidiosus). These initial investigations into the phylogenetic relationships of putative members of the genus Myxobolus clearly indicate the potential limitations of groupings based on size and morphological properties of the spores and host species infected. We propose that 18S rRNA gene sequences, combined with information on tissue tropism, host species infected, and developmental cycles in the fish and alternate host (when and if known) be given greater consideration in taxonomic placements of myxosporeans.

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations.

Abstract: A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificites of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions.

Pcr detection and identification of leishmania parasites in clinical specimens in ecuador: a comparison with classical diagnostic methods

Abstract: A simplified polymerase chain reaction (PCR)-based assay was used for detection and typing of Leishmania parasites in clinical specimens from patients suspected of cutaneous leishmaniasis. Using cultures as the reference standard, our PCR detection method was more sensitive (92%) than classical diagnostic techniques, including microscopy (42% sensitivity), histologic staining (33%), and IgG enzyme-linked immunosorbent (20%). The PCR assay was also 100% specific. Parasites in both lesion biopsies and isolates cultured from lesion aspirates were identified as Leishmania braziliensis by PCR. In this study, we have demonstrated the suitability of simplified PCR assays for the simultaneous diagnosis and typing of parasites causing cutaneous leishmaniasis in a developing country where leishmaniasis is endemic.

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula.

Abstract: Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.

Genotyping of Toxoplasma gondii associated with abortion in sheep.

Abstract: Genotypes of Toxoplasma gondii in naturally infected tis- sues from 13 ovine abortions from 10 farms in the United Kingdom, from 2 wild rodents captured on 1 farm, and from 2 isolates in labo- ratory mouse brains (made from the hearts of healthy, infected lambs) all conformed to the type II lineage determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) anal- ysis of the SAG2 gene. PCR was applied directly to tissue extracts without parasite isolation or DNA extraction. These results demonstrate the feasibility of direct analysis of T. gondii lineages from samples derived either from clinical cases or asymptomatic infections. The populations of several protozoan parasites appear to be made up of a limited number of genetically similar lineages (Tibayrenc et al., 1990), each propagated by a predominance of asexual replication. In a number of protozoans, certain geno- types exist that can often be associated with particular hosts and diseases (Tibayrenc, 1993). The protozoan, Toxoplasma gondii is a zoonotic obligate intracellular parasite with a global distribution that infects man and a wide variety of birds and mammals including the Felidae, which serve as hosts to the sexual phase of replication (Dubey and Frenkel, 1972). Trans- mission of T. gondii occurs when susceptible hosts ingest oo- cysts shed in cat feces or by ingestion of T. gondii tissue cysts in chronically infected tissues (Beverley, 1976) and, in some cases, by vertical transmission (Beverley, 1959; De Roever- Bonnet, 1969; Owen and Trees, 1998). There are many recog- nized strains of T. gondii that differ in certain biological prop- erties but that are all similar antigenically and morphologically, and hence a single species is recognized (Sibley and Boothroyd, 1992). By isoenzyme electrophoresis, restriction fragment length polymorphism (RFLP) analysis, and random-amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR), the species has been subdivided into 2 or 3 major groupings

Structure, biodiversity, and historical biogeography of nematode faunas in holarctic ruminants: morphological and molecular diagnoses for Teladorsagia boreoarcticus n. sp. (Nematoda: Ostertagiinae), a dimorphic cryptic species in muskoxen (Ovibos moschatus).

Abstract: Discovery of the ostertagiine nematode Teladorsagia boreoarcticus n. sp. in muskoxen, Ovibos moschatus, from the central Canadian Arctic highlights the paucity of knowledge about the genealogical and numerical diversity of nematode faunas characteristic of artiodactyls at high latitudes across the Holarctic. Teladorsagia boreoarcticus is a dimorphic cryptic species distinguished from Teladorsagia circumcincta/Teladorsagia trifurcata in domestic sheep by a 13% divergence in the ND4 region of mitochondrial DNA, constant differences in the synlophe, and significantly longer esophageal valve, spicules, gubernaculum, and bursa. Teladorsagia boreoarcticus represents an archaic component of the North American fauna and may have a Holarctic distribution in muskoxen and caribou. Recognition of T. boreoarcticus in muskoxen, in part, corroborates hypotheses for the existence of a cryptic species complex of Teladorsagia spp. among Caprinae and Cervidae at high latitudes and indicates the importance of climatological determinants during the late Tertiary and Pleistocene on diversification of the fauna. Also reinforced is the concept of the North American fauna as a mosaic of endemic and introduced species. Discovery of a previously unrecognized species of Teladorsagia has additional implications and clearly indicates that (1) our knowledge is incomplete relative to potentially pathogenic nematodes that could be exchanged among domestic and wild caprines; (2) we do not have sufficient knowledge of the fauna to understand the ecological control mechanisms (limitations) on dissemination and host range; and (3) an understanding of historical and geographical influences on the genealogical diversity and distribution of nematode faunas in domestic and wild ruminants is requisite to define the interface between agricultural and natural ecosystems across the Holarctic.

Evolution of the Major Lineages of Tapeworms (Platyhelminthes: Cestoidea) Inferred From 18S Ribosomal DNA and Elongation factor-1alpha

Abstract: The interrelationships of the tapeworms (Platyhelminthes: Cestoidea) were inferred by analysis of complete gene sequences (approximately 2,200 bp) of 18S small subunit ribosomal DNA (18S) and partial gene sequences (approximately 900 bp) of elongation factor-1alpha (Ef-1alpha). New collections were made of 23 species representing each of the 14 currently recognized orders of tapeworms, including the Amphilinidea, Gyrocotylidea, and the 12 orders of the Eucestoda. Sequences were determined directly from polymerase chain reaction (PCR) products by either manual or automated methods. Nucleotide sequences of platyhelminth species outside of the Cestoidea were obtained for rooting the resulting trees. The 18S sequences were aligned with reference to the secondary structural features of the gene and the Ef-1alpha sequences were aligned with reference to their corresponding amino acid residues. Significant length variation among taxa was observed in the V2, V4, and V7 variable regions of the 18S gene. Such positions where sequences could not be aligned confidently were excluded from the analyses. Third codon positions of the Ef-1alpha gene were inferred to be saturated at an ordinal level of comparison. In addition, a short (approximately 35 bp) intron region of the Ef-1alpha gene was found to be shared only among the eucestode taxa, with the exception of Spathebothrium simplex (Spathebothriidea), which lacked the intron. Complete alignments showing structural features of the genes and sites excluded from analysis are provided as appendices. The sequence data were partitioned into 7 data sets in order to examine the effects of analyses on different subsets of the data. Analyses were conducted on the 2 genes independently, different codon positions of Ef-1alpha, amino acid sequences of Ef-1alpha, and combinations thereof. All subsets of the data were analyzed under the criterion of maximum parsimony as well as minimum evolution using both maximum-likelihood estimated, and LogDet-transformed distances. Results varied among the different data partitions and methods of analysis. Nodes with strong character support, however, were consistently recovered, and a general pattern of evolution was observed. Monophyly of the Cestoidea (Amphilinidea + Gyrocotylidea + Eucestoda) and Eucestoda and the traditionally accepted positions of the Amphilinidea and Gyrocotylidea as sister lineages to the Eucestoda were supported. Within the Eucestoda, the Spathebothriidea was found to be the sister of all other eucestodes. The remaining orders generally formed a diphyletic pattern of evolution consisting of separate difossate and tetrafossate lineages. This pattern was not universally observed among the analyses, primarily because the trypanorhynch and diphyllidean taxa showed instability in their phylogenetic position. Additional relationships that showed high levels of nodal support included a sister relationship between the Pseudophyllidea and Haplobothriidea and a clade uniting the Cyclophyllidea, Nippotaeniidea, and Tetrabothriidea. The Tetraphyllidea, as currently defined, was found to be paraphyletic without the inclusion of the orders Proteocephalidea and, possibly, Lecanicephalidea. Ordinal status of a monophyletic Litobothriidea, currently classified within the Tetraphyllidea, was found to be supported from a phylogenetic perspective.

Phylogeny of the leech family Glossiphoniidae based on mitochondrial gene sequences and morphological data.

Abstract: The phylogenetic relationships of the Glossiphoniidae (Rhynchobdellida) were investigated using morphological characters and the mitochondrial genes cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit 1. Thirty-five taxa representing 10 of the 23 currently recognized glossiphoniid genera were sampled, including more than 70% of known North American species, as well as others from Europe, South America, Africa, and a species endemic to Lake Baikal. Outgroup taxa included species from the Piscicolidae and Ozobranchidae. Cladistic analysis resulted in 1 most-parsimonious tree. Subfamily distinctions, i.e., Haementeriinae, Theromyzinae, and Glossiphoniinae, that have been based on eye morphology and reproductive biology are not corroborated. Results also provide insights into several problematic genus-level classifications. For example, relationships of Placobdella and Haementeria are clarified and elimination of Desserobdella may be necessary. Bloodfeeding from vertebrates is seen to be a primitive characteristic that has been lost twice within the clade. The hypothesis that the biannulate leech, Oligobdella biannulata, represents an important transitional form is re-evaluated in a phylogenetic context.

A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine

Abstract: A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P < 0.05, Wilcoxon sign rank test). There was a positive correlation (Spearman's p = 1.0, P < 0.0001) between the change in prevalence in juvenile cats and the change in prevalence in finishing pigs. The seropositivity rate (MAT ≥ 1:25) in mice among all farms decreased from 4% in 1992-1993 to 0% in 1996. The mean prevalence of T. gondii tissue cyst isolation for mice on all farms decreased from 1.1% in 1994, to 0.8% in 1995, and to 0.5% in 1996. The results of this study suggest that the reduced exposure of pigs to T. gondii was due to the administration of the T. gondii vaccine to cats.

Cryptosporidium sp. and Giardia sp. infections in mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda.

Abstract: For conservation purposes and because of growing ecotourism, some mountain gorilla (Gorilla gorilla beringei) populations have been habituated to humans. Fecal specimens (n = 100) of nonhabituated and human-habituated gorillas (5 populations; 6 age classes) were tested for Cryptosporidium sp. oocysts and Giardia sp. cysts by conventional staining and immunofluorescent antibody (IFA). Cryptosporidium sp. infections (prevalence 11%) were not restricted to very young gorillas but were observed in 3-yr-old to >12-yr-old gorillas; most of the infections (73%) occurred in human-habituated gorillas. The prevalence of Giardia sp. infections was 2%; 1 nonhabituated gorilla was concomitantly infected. Oocysts of Cryptosporidium sp. in the gorilla stools were morphologically, morphometrically, and immunologically undistinguishable from a bovine isolate of Cryptosporidium parvum oocysts. Mean concentration of Cryptosporidium sp. oocysts and Giardia sp. cysts in gorilla stools was 9.39x10(4)/g, and 2.49x10(4)/g, respectively. There was no apparent relationship between oocyst concentration and gorilla age, sex, or habituation status. Most Cryptosporidium sp. infections found in gorillas with closest proximity to people may be a result of the habituation process and ecotourism. This study constitutes the first report of Cryptosporidium sp. infections in the family Pongidae, in the free-ranging great apes, and in the species of gorilla.

Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species.

Abstract: Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.

The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Abstract: Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level

Cryptosporidium parvum: structural components of the oocyst wall.

Abstract: Cryptosporidium parvum, an enteropathogenic parasite, infects a wide range of mammals including man and constitutes a substantial veterinary and medical threat due to its ubiquitous distribution and the stability of the oocyst stage. The oocyst wall of C. parvum is known to be extremely resistant to chemical and mechanical disruption. Isolated oocyst walls are shown by both thin sectioning and negative staining transmission electron microscopy to possess a filamentous array on the inner surface. This filamentous array can be greatly depleted by digestion with proteinase K and trypsin, but pepsin has less effect. Ultrasonication of the untreated oocyst walls produced almost no fragmentation, but extension of the suture resulted in inward spiraling of the wall to generate ellipsoid and cigar-shaped multilayer bodies, with the filamentous array still present. When ultrasonicated, proteinase K-digested oocyst walls progressively fragmented into small sheets. These wall fragments, depleted of filaments, are shown by negative staining to possess a pronounced linearity, indicative of an integral highly complex lattice structure.

Genetic diversity and recruitment pattern of Schistosoma mansoni in a Biomphalaria glabrata snail population: a field study using random-amplified polymorphic DNA markers.

Abstract: Random-amplified polymorphic DNA markers have been used to assess the amount and the distribution of the genetic diversity of Schistosoma mansoni within a natural population of Biomphalaria glabrata at a transmission site of the murine schistosomiasis focus of Guadeloupe. Despite high infection rate and heavy schistosome load within the definitive hosts (Ratus rattus), prevalences within intermediate snails ranged from 0.2 to 4.8%. Whatever the transmission season may be (rainy vs. dry), most of the infected snails were spatially aggregated and 88.4% of them harbored a single parasite genotype indicative of a monomiracidial infection; 4.7% had dual sex infections and a parasite intensity not exceeding 3 miracidia per snail. A substantial resistance level toward the parasite and recruitment regulatory process within snails may explain in part the observed low parasite prevalences and intensities. Considering such a distribution pattern of larval S. mansoni genetic diversity among B. glabrata, mobility of the definitive hosts, or rapid turnover of infected snails, or both, are required to maintain genetic heterogeneity within adult schistosome populations.

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination.

Abstract: Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.

Combined ribosomal DNA and morphological analysis of individual gyrodactylid monogeneans.

Abstract: A method is presented for the isolation and analysis of hamuli, marginal hooks, and bars from individual gyrodactylid monogeneans using scanning electron microscopy (SEM), while simultaneously processing parasites for rDNA analysis using the polymerase chain reaction (PCR). The haptors of ethanol-fixed gyrodactylids were protease digested to liberate hooks for SEM, whereas DNA extracted from the bodies was used for PCR. The method resulted in hooks and hamuli being prepared from more than 90% of Gyrodactylus turnbulli individuals, a significant improvement on previously published digestion-based SEM techniques. PCR on the same parasites was less successful, but sequence data were obtained from 50% of individuals. Amplification of rDNA internal-transcribed spacer regions from individual worms used for SEM gave PCR products consistent with those predicted from our previous sequence analysis. This method allows the correlation of morphology and DNA sequence from the same individual and can be applied to ethanol-fixed material, such as field collected and museum specimens.

Serological differences in Neospora caninum-associated epidemic and endemic abortions.

Abstract: A sandwich enzyme-linked immunosorbent assay (ELISA) for the sensitive and specific detection of bovine antibodies to Neospora caninum was developed and evaluated using sera from cattle experimentally infected with N. caninum. Toxoplasma gondii. Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis hirsuta, Eimeria bovis, Cryptosporidium parvum, Babesia divergens. and field sera from naturally exposed animals. Field sera were classified using a gold standard that included the results from an indirect fluorescent antibody rest (IFAT) and an immunoblot (IB). Based on these gold standard results, i.e., IFAT-IB results, an equal relative sensitivity and specificity of 94.2% (theta(0),) was reached when a cutoff of 0.034 (d(0))was employed. The analysis of IFAT-IB-positive held sera showed that within groups of aborting and nonaborting dams, the animals from herds with endemic N. caninum-associated abortions had significantly higher ELISA indices than animals from herds with N. caninum-associated, epidemic abortions. By contrast, IFAT-IBpositive aborting dams from herds with endemic N. caninum,-associated abortions had significantly lower IFAT titers than IFAT-IBpositive abetting dams from herds with epidemic N. caninum-associated abortions. This is the first time that statistically significant serological, differences between herds exhibiting epidemic and endemic N. canimum-associated abortions are described

Life history, pathology, and description of Kudoa ovivora n. sp. (Myxozoa, Myxosporea): an ovarian parasite of Caribbean labroid fishes.

Abstract: We describe Kudoa ovivora n. sp. from ovaries of bluehead wrasse, Thalassoma bifasciatum, and record its presence in 6 species (Labroidei) collected in the San Blas Islands. Panama. Kudoa ovivora spores are quadrate with rounded edges in apical view, oval-shaped with apical valve extensions in side view (mean spore dimensions: length 6.5 microns, width 7.7 microns, thickness 6.9 microns; mean polar capsule dimensions: length 2.1 microns, width 1.5 microns). This is the first Kudoa species from gonads of fishes. Prevalence of infection varied among labrids (Thalassoma bifasciatum, Halichoeres bivittatus, Halichoeres garnoti, Halichoeres poevi), with T. bifasciatum exhibiting the greatest prevalence. Density of infection, measured as percent infected eggs, also varied among species with highest densities occurring in H. garnoti. Kudoa ovivora may not require an intermediate host because fishes fed infected tissue developed more infections than unfed fish. Infected eggs are inviable and larger and heavier than uninfected eggs. Infected eggs contain more organic and inorganic material, indicating that K. ovivora increases resource allocation to eggs. Therefore, infected females may have reduced growth, fecundity, and/or spawning activity. Because males were uninfected and all identified hosts are protogynous sequential hermaphrodites, further studies of K. ovivora may provide new insights on the costs/benefits of sex change.

Comparison of the internal transcribed spacer, ITS-1, from Sarcocystis falcatula isolates and Sarcocystis neurona.

Abstract: The genetic diversity among 6 Sarcocystis falcatula iso- lates derived from geographically distinct regions in the U.S.A. was detected using the first internal transcribed spacer region 1 (ITS-1) of the rRNA gene. These sequences were then compared to the full se- quence from a Sarcocystis neurona isolate obtained from a California horse diagnosed with equine protozoal myeloencephalitis. No nucleo- tide differences were detected over partial sequence analysis of 2 ad- ditional S. neurona isolates; however, the complete nucleotide sequence for the ITS-1 region was not compared. Twelve nucleotide differences were consistently detected when aligned sequences of S. neurona were compared to those of the S. falcatula isolates. Additional nucleotide base changes were detected among the S. falcatula isolates, but these changes were not consistent in all the S. falcatula isolates. These results indicate that S. falcatula may be comprised of a heterogeneous popu- lation and that the ITS-1 region can be used to distinguish S. neurona from S. falcatula used in this study. ABSTRACT: The genetic diversity among 6 Sarcocystis falcatula iso- lates derived from geographically distinct regions in the U.S.A. was detected using the first internal transcribed spacer region 1 (ITS-1) of the rRNA gene. These sequences were then compared to the full se- quence from a Sarcocystis neurona isolate obtained from a California horse diagnosed with equine protozoal myeloencephalitis. No nucleo- tide differences were detected over partial sequence analysis of 2 ad- ditional S. neurona isolates; however, the complete nucleotide sequence for the ITS-1 region was not compared. Twelve nucleotide differences were consistently detected when aligned sequences of S. neurona were compared to those of the S. falcatula isolates. Additional nucleotide base changes were detected among the S. falcatula isolates, but these changes were not consistent in all the S. falcatula isolates. These results indicate that S. falcatula may be comprised of a heterogeneous popu- lation and that the ITS-1 region can be used to distinguish S. neurona from S. falcatula used in this study.

Acanthamoeba strains isolated from organs of freshwater fishes

Abstract: Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.

Sarcocystis speeri N. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana).

Abstract: The North American opossum (Didelphis virginiana) is host to at least 3 species of Sarcocystis: Sarcocystisfalcatula, Sarcocystis neurona, and a recently recognized Sarcocystis sp. A new name, Sarcocystis speeri, is proposed for the third unnamed Sarcocystis. Immunodeficient mice are an experimental intermediate host for S. speeri. Sarcocystis speeri sporocysts are 12-15 x 8-10 microm in size, and its schizonts are found in many organs of mice. Sarcocysts of S. speeri are found in skeletal muscles and they are up to 5 mm long and filiform. By light microscopy, the sarcocyst wall is thin (<1 microm thick); ultrastructurally, the cyst wall is up to 1.8 microm thick and has characteristic steeple-shaped villar protrusions surmounted by a spire. Sarcocystis speeri schizonts are morphologically and antigenically distinct from schizonts of S. neurona, and S. speeri sporocysts were not infective to budgerigars (Melopsittacus undulatus).

Prevalence of antibodies to Neospora caninum in horses in North America.

Abstract: Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.

Infection and immunity with the RH strain of Toxoplasma gondii in rats and mice.

Abstract: Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.

Multispecies Plasmodium infections of humans.

Abstract: We analyzed point-prevalence data from 19 recent studies of human populations in which either Plasmodium ovale or Plasmodium vivax co-occur with Plasmodium falciparum and Plasmodium malariae. Although the only statistical interactions among, sympatric congeners are pairwise, the frequencies of mixed-species infections relative to standard hypotheses of species sampling independence show no strong relation to overall malaria prevalence. The striking difference between the P. falciparum-P. malariae-P. ovale and the P. falciparum-P. malariae-P. vivax data is that the first typically shows a statistical surplus of mixed-species infections and the second a deficit. This suggests that the number of Plasmodium species present in a human population may be less important in determining the frequencies of mixed-species infections than is the identity of those species.

Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (Aminosidine) and gentamicin.

Abstract: Cutaneous leishmaniasis is presently treated with 20 days of parenteral therapy with a frequently toxic drug (antimony). Topical formulations of paromomycin (15%) plus methylbenzethonium chloride (MBCL, 12%) or plus urea (10%) in soft white paraffin have been tested for Old and New World disease in humans. We compared the efficacy of a new topical formulation, WR 279,396 (paromomycin [15%] plus gentamicin [0.5%]) to the clinical formulations in the treatment of cutaneous disease in BALB/c mice. Sixty-day-old lesions were treated twice a day for 10 days, and the response to therapy was determined over a further 70 days. For ulcers due to Leishmania major or to Leishmania mexicana, 100% of lesions in the WR 279,396 group healed by day 20 after therapy and did not relapse by day 70; 83% of lesions healed without relapse in the paromomycin-MBCL group. In the paromomycin-urea group, 100% of L. major lesions healed by day 30 but 30% relapsed. For ulcers due to Leishmania panamensis or Leishmania amazonensis, all lesions treated with WR 279,396 healed and did not relapse; < 50% of lesions treated with paromomycin-MBCL healed by day 30, and all lesions relapsed by day 70. In addition to being active, WR 279,396 was not toxic in this model and appears to have a cosmetic effect (promoting hair growth, healing, and limiting the size of the scar).

A cladistic approach to relationships in pentastomida.

Abstract: The positioning of the Pentastomida among the Metazoa has provoked many debates up to the present. On the other hand, the internal relationships among the pentastomid subgroups have received much less attention in the past. We provide the first phylogenetic analyses under Hennigian principles. Thirty-two morphological characters were selected from the primary literature, analyzed manually, and then with the program Hennig86. Four most parsimonious trees were obtained; these were analyzed by successive weighting and reduced to 1 consensus cladogram 380 steps long, with a consistency index of 0.98 and a retention index of 0.99. Characters were also analyzed as unordered, producing results that were congruent with the previous analyses. The internal groups were ordered according to the following system: (Heymonsicambria + Haffnericambria + Bock- lericambria (Cephalobaenida (Railietiellida nov. (Reighardiida nov. (Porocephalida (Linguatuloidea (Linguatulidae + Subtrique- tridae) + Porocephaloidea (Sebekidae + Porocephalidae))))))). This phylogenetic system is largely congruent with the first modern taxonomic arrangement proposed for the Pentastomida. Pentastomida is a group of endoparasites that infest the re- spiratory tract of vertebrates, mainly reptiles. The -131 de- scribed species are broadly distributed, chiefly in tropical re- gions. Despite their extreme specializations for a parasitic way of life, different combinations of characters have been used to suggest relationships with Cestoda (Frohlich, 1789), Trematoda (Humboldt, 1811), Crustacea (Beneden, 1849; Wingstrand, 1972; Abele et al., 1989), Nematoda (Diesing, 1850), Chelicer- ata (Leuckart, 1860; Sambon, 1922a), Annelida (Heymons, 1935), Tardigrada and Onychophora (Cuenot, 1949), and Myr- iapoda (Osche, 1963). We may distinguish 5 periods in the systematic history of the Pentastomida. The first begins with the first species descrip- tions, culminating with the proposal of the family Linguatulidae (Leuckart, 1860). This taxon grouped all the known species of pentastomids. The second period is represented mainly by the system of Sambon (1922a, 1922b). The Pentastomida were arranged by their morphology, hosts, and geographical distribution. All spe- cies were placed in the single family Linguatulidae, within the order Acarina. The Railietiellinae, with hooks placed on lobes, with the female pore placed anteriorly, and with a sacculiform uterus, contained the genera Raillietiella and Reighardia. The genus Cephalobaena, previously described by Heymons (1922), was not accepted by Sambon (1922a, 1922b) who considered it a synonym of Raillietiella. The Porocephalinae, with sessile hooks, a posterior female pore, and a long and contorted tubular uterus, was subdivided into 3 sections: Sebekini (containing Se- bekia, Alofia, Leiperia, Sambonia, and Diesingia), Porocephal- ini (containing Porocephalus, Waddycephalus, Kiricephalus, and Armillifer), and Linguatulini (containing Linguatula and Subtriquetra). with Porocephalus, Kiricephalus, Armillifer, Waddycephalus, and Ligamifer; Sebekinae, with Sebekia, Alofia, and Leiperia; Diesinginae, with Diesingia and Elenia; and Samboninae, with Sambonia). The fourth period includes the proposal of Fain (1961), whose subdivision of Porocephalida into 2 suborders (Poroce- phaloida and Linguatuloida) was not generally accepted, but on the other hand his suggestion that Subtriquetra (now Subtri- quetridae) be separated from the Linguatulidae has been largely followed.

Differences in the second internal transcribed spacer (ITS-2) of eight species of gastrointestinal nematodes of ruminants.

Abstract: Genetic differences in the nucleotide sequence of the second internal transcribed spacers (ITS-2) among Trichostrongylus axei, Trichostrongylus colubriformis, Ostertagia ostertagi, Cooperia oncophora, Cooperia punctata, Nematodirus helvetianus, Nematodirus filicollis, and Haemonchus contortus are described. The ITS-2 sequences of the 8 species ranged between 230 and 241 base pairs in length. Sequence similarities between the different genera varied between 60% and 80%. Identities between the different species within a genus varied between 99% for C. oncophora and C. punctata, 95% for T. axei and T. colubriformis, and 89% for N. helvetianus and N. filicollis. The ITS-2 sequences proved to be useful for species differentiation. Except for the species of Cooperia (2.07% intraspecific variations for C. oncophora and 0.83% for C. punctata) the degree of intraspecific variations (N. filicollis 0.85%, T. colubriformis 1.26%, T. axei 1.27%, H. contortus 2.60%, O. ostertagi, and N. helvetianus no variation) was markedly lower than the interspecific variations allowing a reliable differentiation within the ITS-2 region between single species.