Showing papers in "Journal of Parasitology in 1995"

Duration of immunity to shedding of Toxoplasma gondii oocysts by cats

Abstract: Cats that have shed Toxoplasma gondii oocysts are considered to be immune to reshedding of oocysts. To investigate if this immunity persists in cats for 6 yr, 12 4-6-mo-old cats without T. gondii antibodies were inoculated orally with tissue cysts of the ME-49 strain (6 cats) and the TS-2 strain (6 cats) of T. gondii. All of them shed > or = 20 million oocysts between 4 and 13 days after feeding tissue cysts. Two cats became ill between 11 and 13 days after primary infection; 1 died on the 13th day, and the other had to be killed on the 11th day because of generalized acute toxoplasmosis. Toxoplasma gondii oocysts were not found on the hair of 10 cats examined 7 days after cats had shed millions of oocysts. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. None of the challenged cats shed oocysts. One cat died due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Four of these 9 cats re-shed T. gondii oocysts; 3 of them had been challenged also at 39 days after primary infection. Two control cats housed together with chronically infected cats for 6 yr remained seronegative for T. gondii; both of these shed oocysts after challenge with the P89 strain.

Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois

Abstract: Field studies were conducted on 47 swine farms in Illinois during 1992 and 1993 to identify sources and reservoirs of Toxoplasma gondii infection Blood samples were obtained from swine and from trapped wildlife Serum antibodies to T gondii were determined using the modified agglutination test, incorporating mercaptoethanol Antibodies to T gondii (titer > 25) were found in 97 of 4,252 (23%) finishing pigs, 395 of 2,617 (151%) sows, 267 of 391 (683%) cats, 126 of 188 (670%) raccoons, 7 of 18 (389%) skunks, 29 of 128 opossums (227%), 6 of 95 (63%) rats, 3 of 61 (49%) white-footed mice (Peromyscus sp), and 26 of 1,243 (21%) house mice (Mus musculus) Brains and hearts of rodents trapped on the farm were bioassayed in mice for the presence of T gondii Toxoplasma gondii was recovered from tissues of 7 of 1,502 (05%) house mice, 2 of 67 (30%) white-footed mice, and 1 of 107 (09%) rats Feces of 274 cats trapped on the farms and samples of feed, water, and soil were bioassayed in mice for the presence of T gondii oocysts Toxoplasma gondii was isolated from 2 of 491 (04%) feed samples, 1 of 79 (13%) soil samples, and 5 of 274 (18%) samples of cat feces All mammalian species examined were reservoirs of T gondii infection All farms had evidence of T gondii infection either by detection of antibodies in swine or other mammalian species, or by detection of oocysts, or by recovery from rodents by bioassay The possibility of transmission of T gondii to swine via consumption of rodents, feed, and soil was confirmed Humans become infected with Toxoplasma gondii usually by ingesting oocysts in food and water contaminated by cat feces or by consuming tissue cysts in undercooked meat (Dubey and Beattie, 1988) Pork is considered to be the most important meat source of T gondii infection in the US (Dubey, 1986) There are potentially serious consequences of T gondii infection in humans Exposure of women to T gondii for the first time during pregnancy can result in perinatal mortality and birth

The demise of a phylum of protists: phylogeny of myxozoa and other parasitic cnidaria

Abstract: The notion that members of the phylum Myxozoa Grasse, 1970 do not properly belong in classifications of protists has frequently been suggested because the infective spores of these parasites are not unicellular. Systematists have failed to be decisive about myxozoan phylogenetic affinities, either finding the suggestion of a cnidarian connection to be preposterous or considering the recent suggestion of a relationship with nematodes to be an obvious failure of molecular phylogenetics. Thus, the group has remained in classifications as a protistan phylum in its own right. The ultrastructure of the development of myxozoans was critically re-examined in order to more fully explore the possibility of morphological synapomorphies with metazoan taxa. These morphological characters, in combination with small ribosomal subunit gene sequences, were used in a phylogenetic analysis in order to assess myxozoan origins. The results unequivocally support the inclusion of myxozoans as a clade of highly derived parasitic cnidarians, and as sister taxon to the narcomedusan Polypodium hydriforme. Reassessment of myxozoans as metazoans reveals terminal differentiation, typical metazoan cellular junctions, and collagen production. Their "polar capsules" are redescribed as typical nematocysts bearing atrichous isorhiza. Insofar as taxa cannot be contained within other taxa of equal rank, the phylum Myxozoa is abandoned and it is recommended that the group as a whole be removed from all protistan classifications and placed in a more comprehensive cnidarian system.

The mechanism of action of avermectins in Caenorhabditis elegans: correlation between activation of glutamate-sensitive chloride current, membrane binding, and biological activity.

Abstract: Xenopus laevis oocytes were injected with mRNA isolated from the free-living nematode Caenorhabditis elegans and the activation and potentiation of a glutamate-sensitive chloride current by a series of avermectin analogs and milbemycin D were determined. There was a strong correlation between the EC50 value determined for current activation in oocytes, the LD95 value for nematocidal activity, and also for the Ki value determined in a [3H]ivermectin competition binding assay. Four of the analogs were tested for potentiation of glutamate-sensitive current and the rank order for potentiation correlated with the EC50 for direct activation of current. We conclude that avermectins and milbemycins mediate their nematocidal effects on C. elegans via an interaction with a common receptor molecule, glutamate-gated chloride channels.

Microevolution and the genetic structure of parasite populations

Abstract: The development of polymerase chain reaction-based methods for assessing the genotypes of small individual organisms will promote groundbreaking investigations of the genetic architecture of parasite populations. Both quantitative genetic models and general knowledge of parasite natural history are useful for making general predictions about the distribution of genetic variation over geographic space. However, designing experimental studies to assess relationships between specific life history variables and patterns of genetic structure in natural populations will be challenging. Traditional biochemical-genetic methods have already been used to study a limited number of parasite populations, and inferred patterns of genetic structure are distinctly different between certain species. Some of these differences in genetic architecture may be explained by parasite or host factors that either promote or retard the dissemination of life cycle stages over geographic space. Many additional empirical studies are needed to characterize basic features of parasite populations, including the spatial distribution and group size of random mating populations and levels of gene flow among parasite subpopulations.

The evolution of virulence: a unifying link between parasitology and ecology

Abstract: For most of the past century, the conventional wisdom in parasitology and epidemiology has been that a well adapted parasite is a benign parasite (parasites being defined broadly to include multicellular, unicellular, and subcellular organisms). This view became firmly entrenched during the second quarter of this century (Smith, 1934; Swellengrebel, 1940). Although objections were raised during the middle decades of this century (Ball, 1943; Cockburn, 1963), they had little impact in the face of the more aesthetically appealing paradigm, which advocated evolution toward balanced, stable associations in which parasites succeeded through peaceful coexistence with their hosts. The problem with this conventional view is that natural selection does not necessarily favor peaceful coexistence. Rather, it favors those genetic instructions that are passed on preferentially as a result of the characteristics they encode. If instructions that cause their bearer to exploit host resources more fully are more successfully passed into future generations as a result of that exploitation, then adaptation to the host will be associated with increased exploitation of the host. Insofar as increased exploitation causes increased harm, evolutionary adaptation to the host will result in increased harm to the host. This conclusion was reached independently by several investigators during the last quarter of the 20th century (Ewald, 1980, 1983; Price, 1980; Levin and Pimentel, 1981; Anderson and May, 1982)--in contrast to the situation during mid-century, this time an alternative theoretical framework grew out of this competing view. Now, at the end of the 20th century this competing paradigm has replaced the "conventional wisdom" among evolutionary biologists. This fundamental change in our view of host/parasite evolution promises to transform perspectives and research programs in parasitology and the health sciences. This transformation, however, is still in progress, largely because of the viscosity at the interface between disciplines. (For a more detailed history of these ideas, see Ewald [1994].) Even now the tenet that well adapted parasites are benign is considered by many to be a guiding principle. Parasitologists still often speak of benignness as a sign of a well adapted parasite or infer that mildness is a sign that a host is a natural host. Similarly, this outdated view has also colored the prevailing interpretations of aspects of infectious diseases, such as the origins and evolution of human immunodeficiency virus (HIV) (Ewald, 1994; Mindell et al., 1995). The inadequacy of this viewpoint can be seen by considering the evolutionary stability of a parasite that is peacefully coexisting with its host. Imagine such a "well adapted" parasite and ask whether a more exploitative variant of that parasite would be more or less successful. If that variant funneled the resources gained from this exploitation into its own propagation, would it pass on more copies of its genetic instructions than the less exploitative variant? For a multicellular parasite this increased exploitation might involve greater conversion of host tissues into parasite eggs or into parasite tissues, which would eventually foster greater reproduction. For a unicellular parasite or a virus, this extra exploitation might involve greater invasion of host tissues, liberating resources for the parasite's replication (as with Shigella), or it might involve production of a toxin that indirectly leads to an increased rate of propagation from a host, e.g., as in Vibrio cholerae or Corynebacterium diphtheriae (Ewald, 1994). If the extra exploitation involves no adverse effects on the host, then the variants that exploited the hosts more fully would win. By this line of reasoning, we expect natural selection to push exploitation at least to the point at which the parasite begins to impose negative effects on the host. The problem now becomes particularly interesting. If one agrees that natural selection could favor parasites that exploited hosts to this point, why not presume that it could favor parasites that exploited hosts even more intensely? Parasite variants that did so would still get the fitness benefits of that exploitation, but they may also incur fitness costs as a result of the negative effects of their exploitation on the host. Indeed, it is this tradeoff that is at the heart of current theory about the evolution of virulence (which I use broadly in this paper to mean the harmfulness of a consumer to the individual it is consuming). Current theory proposes that natural selection may favor even high levels of exploitation by parasites if the negative effects on the host have relatively small negative effects on parasite transmission. Countering these effects, of course, are the evolutionary responses of the host to the parasite. Hosts that reduce a parasite's exploitation leave more of their instructions. The end result is quite different from peaceful coexistence-it is an arms race between parasite and host, which is most intense among those parasites for which natural selection favors intense exploitation. This conclusion leads to an important question: What are the factors that drive some parasites toward intense exploitation of hosts and others toward mild coexistence? In this paper, I address this question and suggest that the answer will provide not only a clearer view of host/parasite coevolution, but of the entire spectrum of consumer associations. The approach promises not only to conceptually unify categories such as predation, parasitism, commensalism, and mutualism, but also to provide insight into the reasons for the evolution of complex life histories among parasites. Studies investigating the evolution of virulence have focused on host mobility because a reduction in mobility is one of the first discernable negative effects at the boundary between benignness and virulence, and because different parasites may depend on host mobility to different degrees. A reduction in host mobility should be especially costly for parasites that rely on host mobility for transmission. Conversely, if transmission is * This paper is based on the R. Barclay McGhee Lecture presented at the 60th Annual Meeting at Fort Collins, Colorado, in August 1994.

Toxoplasma gondii in Iowa sows: comparison of antibody titers to isolation of T. gondii by bioassays in mice and cats.

Abstract: Hearts of 1,000 pigs killed at an abattoir in Iowa were bioassayed for the prevalence of tissue cysts of Toxoplasma gondii. One hundred grams of cardiac muscle from each pig was homogenized, digested in pepsin solution, and bioassayed in 10 mice. Five hundred grams of heart tissue from each of a subset of 183 pigs was also bioassayed in cats. Serum collected from the heart from each pig was assayed for anti-T. gondii antibodies in the modified agglutination test using formalin-fixed whole tachyzoites. Anti-T. gondii antibodies were found in 22.2% of pigs. Viable T. gondii was isolated from a total of 170 pigs; from 50 hearts by bioassay in mice, from 58 hearts by bioassay in both mice and cats, and from 62 pigs by bioassay in cats only. The success of isolation in cats (65.6%) was approximately twice that in mice (31.7%). Percentage of isolations of T. gondii with respect to reciprocal antibody titers (in parentheses) in pigs was: 3.7% ( or = 2,000 to 16,000).

Identification of opossums (Didelphis virginiana) as the putative definitive host of Sarcocystis neurona.

Abstract: Sarcocystis neurona is an apicomplexan that causes equine protozoal myeloencephalitis (EPM) in North and South America. Horses appear to be an aberrant host, because the merozoites continually divide in the central nervous system, without encysting. The natural host species has not previously been identified. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was compared to those of Sarcocystis muris, Sarcocystis cruzi, Toxoplasma gondii, and Cryptosporidium parvum to identify a unique region suitable for a species-specific amplification primer. The S. neurona SSURNA primer was used in a polymerase chain reaction (PCR) assay for the purpose of identifying this organism in feces and intestinal digest of wildlife specimens. Sporocysts were isolated from 4 raccoons (Procyon lotor), 2 opossums (Didelphis virginiana), 7 skunks (Mephitis mephitis), 6 cats (Felis catus), 1 hawk (Accipiter sp.), and 1 coyote (Canis latrans). The S. neurona SSURNA PCR assay and a control PCR assay using protist-specific primers were applied to all sporocyst DNA samples. All sporocyst DNA samples tested positive on the control assay. The SSURNA PCR assay yielded a 484-bp product only when applied to opossum samples. The SSURNA gene of both opossum sporocyst samples was sequenced to determine its relationship to the S. neurona SSURNA gene. The sequence had 99.89% similarity with S. neurona. This suggests that opossums are the definitive host of S. neurona.

Risk factors for transmission of Toxoplasma gondii on swine farms in Illinois.

Abstract: Two epidemiologic studies of risk factors for transmission of Toxoplasma gondii to swine were conducted for farms in Illinois. The first study was a cross-sectional survey of swine farms from the state of Illinois pseudorabies testing program, in which farm owners or managers were interviewed by telephone regarding presence of risk factors for transmission of T. gondii on the farm. There were 123 farms surveyed that provided blood samples for at least 30 sows. The mean sow seroprevalence was 19.5% (median = 10.0%). Multiple regression analysis of the association of sow seroprevalence with outdoor housing of sows, cat access to sow areas, number of sows, open feed storage and water delivery, delayed removal of carcasses, and presence of rodents on the farm indicated that higher sow seroprevalence was associated with cat access to sows (P = 0.009) and fewer sows in the herd (P = 0.05). The second study was a field investigation of 47 swine farms (37 from the cross-sectional study). Data collection included obtaining blood samples from swine, cats, and rodents, and fecal samples from cats, heart and brain tissue from rodents, and feed, water, and soil samples for T. gondii examination. The risk of T. gondii transmission from cats and rodents to sows and finishing pigs was evaluated, taking into account housing conditions and herd size. Multiple regression analysis indicated that T. gondii seroprevalence in finishing pigs increased with more seropositive juvenile cats on the farm (P < 0.0001) and higher seroprevalence in house mice (P = 0.0023).(ABSTRACT TRUNCATED AT 250 WORDS)

Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.

Abstract: In vitro production of Schistosoma mansoni daughter sporocysts (DS) from miracidium-derived mother sporocysts (MS) was achieved by synxenic larval cultivation with cells of the Biomphalaria glabrata embryonic (Bge) cell line. The in vitro growth and viability of MS cocultured with Bge cells or in Bge cell-conditioned medium were significantly extended beyond that of larvae cultured in fresh medium alone. However, complete DS development and emergence from MS were achieved only in the presence of Bge cells. Introduction of either miracidia or previously transformed MS onto Bge cell monolayers resulted in an initial attachment of cultured cells to sporocysts, followed by a gradual encapsulation of larvae by multiple layers of Bge cells. Sporocysts and their encapsulating cells eventually formed large cellular aggregates, within which MS increased 4-fold in size during the first 20 days of cultivation. The timing of in vitro DS development was somewhat variable; however, in general, early embryo formation, i.e., germ cell aggregates with surrounding primitive epithelium, was first detected at 15-20 days of culture, whereas motile, intra-MS daughter stages were seen at 25-30 days and thereafter. Mature, first generation DS, measuring 136 +/- 46 microns long by 22 +/- 6 microns wide, emerged from MS starting at approximately 30-45 days of initial cultivation. Although the basic morphology and size of emergent, in vitro-derived DS were comparable to those propagated in vivo, there was a large reduction in the in vitro reproductive capacity of the MS and a delay in DS culture development.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnostic and prognostic value of K39 recombinant antigen in Indian leishmaniasis.

Abstract: The recombinant product (rK39) of the 39 amino acid repeats encoded by a kinesin-like gene of visceral Leishmania spp. was further evaluated by enzyme-linked immunosorbent assay (ELISA) for its diagnostic potential in Indian kala-azar (VL) and post kala-azar dermal leishmaniasis (PKDL). Anti-rK39 antibodies were highly positive in 20 symptomatic cases, including 6 resistant to single or double chemotherapy, but became negligible or absent in 9 recently cured patients. Endpoint titration of samples from the 20 active cases showed that the anti-rK39 IgG titers fell within a wide range of 10(-2) to > 10(-6), and that their mean was > 1 order of magnitude higher than in VL reported previously. The anti-rK39 IgG titers were correlated with parasite burden found in the patients and remained undiminished in those refractory to chemotherapy. These results indicate that: (1) the K39 epitope is conserved in Indian strains of Leishmania donovani, (2) the extremely high levels of K39 antibodies in both VL and PKDL suggest the application of rK39 for sensitive and specific serodiagnosis, and (3) rK39 ELISA is also valuable for prognostic evaluation of both diseases.

Long-term antibody responses of cats fed toxoplasma gondii tissue cysts.

Abstract: As part of a long-term study on immunity to oocyst shedding, 12 4-6-mo-old cats were inoculated orally with tissue cysts of the ME-49 strain (6 cats) or the TS-2 strain (6 cats) of Toxoplasma gondii. Two cats fed the ME-49 strain died or were killed because of acute toxoplasmosis 12 and 13 days after inoculation (DAI), respectively. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. One cat died following rechallenge infection due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Blood samples were obtained weekly or monthly and sera were analyzed for antibodies to T. gondii using the modified agglutination test (MAT), the Sabin-Feldman dye test (DT), and the enzyme-linked immunosorbent assay (ELISA) for IgM (IgM-ELISA) or IgG (IgG-ELISA). The MAT was performed using both formalin-fixed (FF) and acetone-fixed (AF) tachyzoites. The MAT (FF) was the most sensitive test; cats seroconverted within 14 DAI and high titers (> 10,000) persisted > 6 yr, although cats had no clinical signs. The MAT titers using the AF detected recent exposure and titers declined sharply after 2 mo postinoculation. DT and ELISA titers were lower and developed slower than MAT titers. Fluctuations in antibody titers were limited to 8-fold during the 6-yr observation period. Anamnestic serum antibody responses were seen in 2 cats after the final challenge, but not after first challenge.

Neobenedenia girellae (Hargis, 1955) Yamaguti, 1963 (Monogenea: Capsalidae) from cultured marine fishes of Japan.

Abstract: The monogenean Neobenedenia girellae (Hargis, 1955) Yamaguti, 1963 is redescribed and reported for the first time in Japan. The parasite was recovered from the body surface, fins, and occasionally from the eyes of 14 species, comprising 5 families of cultured marine fishes from several localities in southwestern Japan. Neobenedenia melleni (MacCallum, 1927) sensu Kaneko et al. (1988) from tilapia (Oreochromis mossambicus) in Hawaii is synonymized with this species. Examination of original specimens (syntypes) of N. melleni sensu MacCallum (1927) revealed differences with N. girellae in having a wide and rounded body, a prominently large anterior hamuli, and absence of glands of Goto. This Neobenedenia from Japanese fishes sometimes showed an unusual morphology of the individual parts of the median sclerites. The potential threat of N. girellae to the health of cultured Japanese fishes is indicated by its low host specificity, wide distribution, and ability to cause mortality due to heavy infection. Unregulated importation of amberjack fry (Seriola dumerili) to Japan appears to be the source of N. girellae infection in Japanese fishes since 1991.

Vertical transmission of Neospora caninum in mice.

Abstract: Herein we report a murine model to examine transplacental transmission, transmammary transmission, or both, of Neospora caninum. Prevalence of transplacental transmission in outbred Swiss-Webster mouse pups was 85%, with 11 of 13 litters containing at least 1 transplacentally infected pup. Sixty-two percent of litters born to experimentally infected dams contained 85% or more transplacentally infected pups. Numbers of pups congenitally infected per litter was higher if dams were inoculated during the first half of pregnancy. Transplacental transmission decreased to 25% when a singly infected dam delivered a second litter. Transmammary transmission was observed in 1 of 51 pups suckling dams experimentally infected 5, 10, or 15 days postparturition. No pups were infected when cross-fostered onto chronically infected dams.

Mouse model for central nervous system Neospora caninum infections.

Abstract: Neospora caninum is a protozoan parasite that causes severe disease in transplacentally infected dogs and abortions in domestic ruminants. Rodent models of neosporosis rely on treatment of hosts with methylprednisolone acetate (MPA) to enhance infections. The present study reports the development of an inbred BALB/c mouse model that results in central nervous system neosporosis in the absence of MPA treatment. Seven of 12 BALB/c mice died 26-70 days after subcutaneous (s.c.) inoculation with tachyzoites of the NC-1 strain of N. caninum, and none of 12 BALB/c mice died after s.c. inoculation with tachyzoites of the NC-3 strain. None of 8 HSD:ICR mice (4 mice, NC-1 strain; 4 mice, NC-3 strain) developed clinical neosporosis or died after s.c. inoculation with tachyzoites. Control BALB/c (2) and HSD:ICR (2) mice s.c. inoculated with Hanks' balanced salt solution did not develop clinical signs of disease. Some mice in all N. caninum-inoculated groups had brain lesions, but significantly (P < 0.05) more BALB/c mice inoculated with the NC-1 strain had brain lesions.

A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry.

Abstract: ABSTRACr: A flow cytometric method for the quantification of Cryptosporidiumparvum oocysts in stool specimens was developed to replace conventional microscopic immunofluorescent assays. Fecal pellets were collected from control (uninfected) severe combined immune-deficient mice, suspended in 2.5% potassium dichromate at a ratio of 400 Ml per pellet, and homogenized by vortexing. Purified oocysts were added to the samples (105, 104, 103, and 102/ml). Aliquots (200 Ml) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradients. The oocyst-containing fractions were collected, washed, and incubated with an oocyst-specific monoclonal antibody (labeled with fluorescein isothiocyanate) for 30 min at 37 C. Sample volumes were adjusted to 600 Al with phosphate-buffered saline and assayed by using logical gating of forward/side scatter and fluorescence signal on a flow cytometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from chronically infected mice demonstrated that the flow cytometry method was approximately 10 times more sensitive than conventional immunofluorescent assays. Cryptosporidium parvum parasite loads in experimentally infected animal models have been determined by semiquantitative and quantitative methods. Examination of histologic sections of intestinal and extraintestinal organs for cryptosporidial life cycle stages is complemented by the enumeration of oocysts in stool samples. Oocyst enumeration methods rely on the concept that a stool represents a sampling of the entire intestinal tract and as such is less subject to the often nonrandom, nonuniform distribution of parasitic life cycle stages across the epithelial cell surfaces of intestinal and extraintestinal tissues. Conventional methods to enumerate oocysts employ microscopic examination of fecal wet mounts, fecal concentrates, stained, or antibody-labeled specimens. Numerous methods have been reported for the isolation, concentration, staining, labeling, and enumeration of oocysts (Arrowood and Sterling, 1987; Kilani and Sekla, 1987; Garcia and Current, 1989; Mead et al., 1991; Sterling and Arrowood, 1993). Many of these methods are subject to operator error or variability caused by differing degrees of technical proficiency at microscopic methodologies. In the present study, we contrast an improved microscopic method of enumerating fecal oocysts with a new method for evaluating Cryptosporidium parasite loads that circumvents the traditional microscopic examination of stool preparations. The new method combines a concentration and purification technique with an optimized flow cytometric technique for enumerating fecal oocysts in experimentally infected mouse models of cryptosporidiosis.

Parasitic and infectious diseases: epidemiology and ecology.

Abstract: M.E. Scott, Populations are Dynamic. G. Smith, So You Want to Write a Model. G. Smith and M.E. Scott, Model Behavior and the Basic Reproduction Ratio. H. McCallum and M.E. Scott, Quantifying Population Processes: Experimental and Theoretical Approaches. G. Smith, Parasite Population Density Is Regulated. G. Smith, Ecological Epidemiology and Standard Measures of Disease Occurence. J.L. Aron and B.A. Silverman, Models and Public Health Applications. D. Wakelin, Host Populations: Genetics and Immunity. N.W. Solomons and M.E. Scott, Nutritional Status of Host Populations Influences Parasitic Infections. V. Apanius and G.A. Schad, Host Behavior and the Flow of Parasites through Host Populations. A.R. McLean, Control of Microparasites through Vaccination. G.F. Medley, Chemotherapy. C. Dye, Vector Control. D.W.T. Crompton, Ascaris lumbricoides. M.E.J. Woolhouse, Epidemiology of Human Schistosomes. B.T. Grenfell and B.M. Bolker, The Population Dynamics of Measles. M.J. Coyne and G. Smith, Trichostrongylid Parasites of Domestic Ruminants. M.G. Roberts and M.A. Gemmell, Echinococcosis. S. Vail, G. Smith, and C. Lord, The Population Biology of Ixodes scapularis, The Vector of Lyme Disease in the Eastern and North Central United States. M.E. Scott and G. Tanquay, Heligmosomoides polygyrus: A Laboratory Model for Direct Life Cycle Nematodes of Man and Livestock. A.P. Dobson and P.J. Hudson, The Population Biology of Trichostrongylus tenuis in Red Grouse, Lagopus lagopus scoticus. G.W. Esch, The Population Biology of the Diplostomatid Trematode, Uvulifer ambloplitis. F. Fenner, Myxomatosis. M.D.B. Burt, The Sealworm Situation. Index.

Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis : implications for reproductive biology, population structure, taxonomy, and epidemiology

Abstract: The nature and extent of genetic variation in Giardia was used to infer its mode of reproduction, population structure, taxonomy, and zoonotic potential. Ninety-seven isolates of Giardia duodenalis, from a defined area in Western Australia and throughout Australia and overseas, were obtained from humans, cats, cattle, sheep, dogs, goat, beaver, and rats. Enzyme electrophoresis revealed extensive genetic variation with 47 different zymodemes. The widespread occurrence of certain zymodemes and the similarity of relationships among isolates inferred from independent genetic markers suggests a clonal population structure for G. duodenalis, although occasional bouts of genetic exchange may occur. The 47 zymodemes clustered similarly in phenetic (UPGMA) and phylogenetic (Fitch-Margoliash) analyses. The level of genetic diversity in isolates from a defined geographical area in Western Australia was similar to the level of diversity in isolates from throughout Australia. These data suggest that clonal lineages within G. duodenalis are evolutionarily independent. Although there was a significant overall correlation between genetic distance separating zymodemes and occurrence in different host species, we found genetically identical isolates from humans and other animals and extensive genetic diversity between isolates from humans. We interpret this as evidence for zoonotic transmission of the parasite.

Sequence analysis and comparison of ribosomal DNA from bovine Neospora to similar coccidial parasites.

Abstract: The nuclear small subunit ribosomal RNA (nss-rRNA) gene sequence of Neospora spp. isolated from cattle was analyzed and compared to the sequences from several closely related cyst-forming coccidial parasites. Double-stranded DNA sequencing of 5 bovine Neospora spp. isolates (BPA1-4), 2 Neospora caninum isolates (NC-1 and NC-3), and 3 Toxoplasma gondii isolates (RH, GT-1, CT-1) were performed and compared to each other, as well as to other sequences available in GenBank for the NC-1 isolate, Sarcocystis muris, and Cryptosporidium parvum. There were no nucleotide differences detected between the Neospora spp. isolates from cattle and dogs. Four nucleotide differences were consistently detected when sequences of Neospora spp. isolates were compared to those of the T. gondii isolates. These results indicate that Neospora spp. and T. gondii are closely related, but distinct, species.

Transmission of Borrelia burgdorferi by Ixodes pacificus nymphs and reservoir competence of deer mice (Peromyscus maniculatus) infected by tick-bite.

Abstract: The transmission of Borrelia burgdorferi to deer mice (Peromyscus maniculatus) by Ixodes pacificus nymphs was investigated experimentally. Deer mice were exposed to infected nymphs for 24, 48, or 72 hr, or until ticks had fed to repletion (> or = 96 hr). Infection status of hosts was assessed 4 wk later by culture of ear-punch biopsies in BSK II medium and by indirect immunofluorescence. Eight mice exposed to ticks for 24 hr did not become Infected. In contrast, infection was acquired by 1 of 9 (11%), 2 of 8 (25%), and 8 of 10 (80%) mice exposed for 48, 72, and > or = 96 hr, respectively. Eight weeks after exposure to infected nymphs, the infectivity of 5 deer mice for I. pacificus larvae was assessed. Overall, 33% of I. pacificus larvae fed on these mice acquired and transstadially passed spirochetes. We conclude that most I. pacificus nymphs require 4 days or longer to transmit spirochetes to deer mice, and that larvae efficiently acquire and maintain spirochetes from mice that have been infected by tick-bite.

Developmentally regulated secretion of cathepsin L-like cysteine proteases by Haemonchus contortus.

Abstract: Cysteine protease activity was present in media collected after 24 hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen, and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with M(r) values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4, but not the L3 larval stage. The active and developmentally regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition, immune evasion, or both.

The epidemiology of ascaris lumbricoides, trichuris trichiura, and hookworm in children in the ranomafana rainforest, madagascar

Abstract: An epidemiological study of intestinal nematodes was conducted with 1,292 children, ranging from birth through 11 yr old, living in the Ranomafana rainforest of southeast Madagascar. Fecal examinations revealed prevalences of 78% for Ascaris lumbricoides, 38% for Trichuris trichiura, 16% for hookworm, and 0.4% for Schistosoma mansoni. Infection intensity was measured indirectly by fecal egg counts and directly by A. lumbricoides expulsion following treatment with pyrantel pamoate. The mean A. lumbricoides worm burden for children, 5-11 yr old, was 19.2 (SD 20.4) worms per child, with a median of 13 worms (n = 428). The distributions were overdispersed for all 3 nematodes. The age profiles showed a rapid acquisition of A. lumbricoides during infancy, increasing to 100% prevalence by age 10. After mebendazole anthelmintic treatment and a 12-mo reinfection period, the nematodes had rebounded to pretreatment prevalence and intensity levels. There was evidence for age-dependent predisposition of the children to infection intensity for each of the 3 nematodes. Dual species intensity correlation was consistently strong for A. lumbricoides and T. trichiura. The significantly higher prevalence and intensity of ascariasis in girls were thought to be related to exposure.

The life history, ultrastructure, and experimental transmission of Hepatozoon catesbianae n. comb., an apicomplexan parasite of the bullfrog, Rana catesbeiana and the mosquito, Culex territans in Algonquin Park, Ontario.

Abstract: Gametogenesis and sporogonic development of a haemogregarine parasite of bullfrogs (Rana catesbeiana) was observed in cells of the Malpighian tubules of laboratory-reared Culex territans that had fed on naturally infected bullfrogs Mature oocysts, which varied considerably in size, were multisporocystic with ellipsoidal sporocysts that contained 4 sporozoites Sporogonic development was completed in about 20 days Mature meronts were observed in the liver and merozoites in erythrocytes of laboratory-reared bullfrogs that had been fed sporocysts 19 days previously Similar attempts to infect laboratory-reared green and northern leopard frogs experimentally were unsuccessful, suggesting rather narrow specificity for this parasite in ranids Gametogenesis and sporogonic stages of this parasite were ultrastructurally similar to those described for Hepatozoon species The parasite appears to be transmitted directly between bullfrogs and mosquitoes in the study area where Cx territans feeds avidly on bullfrogs, which in turn were observed to naturally ingest these mosquitoes Based on data presented in this study and the earlier description by Stebbins in 1903, the haemogregarine parasite of bullfrogs was designated as a new combination, Hepatozoon catesbianae

A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica

Abstract: A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using 1 microgram of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 microgram of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot-blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a dilution series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as "dilution endpoint" PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification of P. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat.

Sarcocystis falcatula from passerine and psittacine birds: synonymy with Sarcocystis neurona, agent of equine protozoal myeloencephalitis.

Abstract: Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis neurona. The horse is a dead-end host for S. neurona and the definitive and intermediate hosts have not previously been identified. We hypothesized that S. neurona is actually Sarcocystis falcatula, a parasite that cycles in nature between Virginia opossums (Didelphis virginiana) and any of a variety of avian intermediate hosts. We extracted DNA from S. falcatula sarcocysts in the muscle of a brown-headed cowbird (Molothrus ater) and from schizonts in a fixed specimen of lung from a Moluccan cockatoo (Cacatua moluccensis). Three segments of the small subunit ribosomal RNA (SSURNA) gene, containing a total of 742 nucleotides, were amplified by the polymerase chain reaction, sequenced, and compared with the SSURNA sequence from two isolates of S. neurona. The S. falcatula sequence was identical to the sequence of the S. neurona isolate UCD-1 and differed in only 3 positions from isolate SN5. Recent evidence, also based on SSURNA sequences, implicates the opossum as the definitive host of S. neurona. Based on the SSURNA gene sequences S. falcatula and S. neurona are synonymous, thus the parasite cycles between opossums and birds maintaining a reservoir of the organism from which horses can be infected.

Myxobolus cultus n. sp. (Myxosporea: Myxobolidae) in the goldfish Carassius auratus transformed from the actinosporean stage in the oligochaete Branchiura sowerbyi.

Abstract: A new myxosporean Myxobolus cultus n. sp. was found in experimentally infected goldfish Carassius auratus exposed to actinosporean spores from the oligochaete Branchiura sowerbyi (Tubificidae). At 2-4 mo following initial exposure, spores of M. cultus were observed in the cartilage of goldfish. A lymphocytic infiltrate surrounded the pseudocysts. Some pseudocysts were destroyed and spores had been engulfed by phagocytes. Phagocytized spores were also found in the epidermis of skin and within melanomacrophage centers in the kidney.

Early Kinetics of Toxoplasma gondii Infection in Mice Infected Orally with Cysts of an Avirulent Strain

Abstract: We determined the early kinetics of Toxoplasma gondii infection in Swiss Webster mice inoculated with the avirulent C strain by counting parasites in the blood, spleen, Peyer's patches, liver, lungs, and brain. Animals were orally inoculated with cysts on day zero (D0), and parasites were counted using a tissue culture method at 12, 24, and 36 hr and 2, 3, 7, 10, 14, 21, 30, 50, and 72 days postinfection. The spleen and Peyer's patches were the first organs found parasitized, on D2 and D3, respectively, followed by the lungs and liver on D7 and the brain on D10. No parasitemia was detected. This suggests that early dissemination of this avirulent strain from the intestine into the general circulation occurs essentially via the lymphatic system. Parasites persisted at a high level in the brain during the chronic phase. In the lungs, parasites were no longer detected by D72, while parasite numbers initially declined in the spleen and Peyer's patches but then showed a second peak, possibly due to recirculation of T. gondii. These results suggest that lymphoid organs play a key role in T. gondii dissemination during the acute phase and may also constitute a persistent source of parasite resurgence.

Trypanosoma cruzi in wild raccoons, opossums, and triatomine bugs in southeast Georgia, U.S.A.

Abstract: The prevalence of Trypanosoma cruzi infection among wild opossums and raccoons live-trapped in 6 southeast Georgia counties was determined. Epimastigotes typical of T. cruzi were observed in liver infusion tryptose medium cultures of blood from 6 of 39 opossums (15.4%) and 12 of 54 raccoons (22.2%). Trypomastigotes (6,000/ml) were observed in a wet mount of blood from 1 of the animals, a raccoon trapped on St. Catherines Island in Liberty County. In addition, T. cruzi parasites were observed in the gut contents of 3 of 5 specimens of the triatomine bug Triatoma sanguisuga (Leconte) collected near human dwellings in Bulloch County. This is the first report of T. cruzi-infected triatomine bugs in Georgia.