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The aggregation of parasites among hosts is associated with differential host exposure and susceptibility to parasites, which varies according to host gender, body size, reproductive status and environmental factors. We evaluated the role of these factors on infestation by Eimeria spp. (Eimeriidae) in the agile gracile mouse opossum (Gracilinanus agilis), a semelparous didelphid inhabiting neotropical savannahs. Eimeria spp. abundance and prevalence among G. agilis were associated with the breeding status of individuals and to a lesser extent to climatic season, with both sexes presenting higher Eimeria spp. burdens during late breeding/wet season. On the other hand, male-biased parasitism was restricted to dry/mating season. We suggest that male spatial organization and diet may account for increased parasite burdens within this sex, although future studies should evaluate the role of physiological differences associated with androgen hormones. Finally, a rapid increase in Eimeria spp. loads among females during the late breeding/wet season seems associated with seasonal changes in susceptibility, due to breeding costs related to semelparity, and exposure to infective propagules, while male-die off seems to explain maintenance of higher Eimeria spp. burdens within this sex in the same period.

Novel Hepatozoon spp. sequences collected from previously unrecognized vertebrate hosts in North America were compared with documented Hepatozoon 18S rRNA sequences in an effort to examine phylogenetic relationships between the different Hepatozoon organisms found cycling in nature. An approximately 500-base pair fragment of 18S rDNA common to Hepatozoon spp. and some other apicomplexans was amplified and sequenced from the tissues or blood of 16 vertebrate host species from the southern United States, including 1 opossum (Didelphis virginiana), 2 bobcats (Lynx rufus), 1 domestic cat (Felis catus), 3 coyotes (Canis latrans), 1 gray fox (Urocyon cinereoargenteus), 4 raccoons (Procyon lotor), 1 pet boa constrictor (Boa constrictor imperator), 1 swamp rabbit (Sylvilagus aquaticus), 1 cottontail rabbit (Sylvilagus floridanus), 4 woodrats (Neotoma fuscipes and Neotoma micropus), 3 white-footed mice (Peromyscus leucopus), 8 cotton rats (Sigmodon hispidus), 1 cotton mouse (Peromyscus gossypinus), 1 eastern grey squirrel (Sciurus carolinensis), and 1 woodchuck (Marmota monax). Phylogenetic analyses and comparison with sequences in the existing database revealed distinct groups of Hepatozoon spp., with clusters formed by sequences obtained from scavengers and carnivores (opossum, raccoons, canids, and felids) and those obtained from rodents. Surprisingly, Hepatozoon spp. sequences from wild rabbits were most closely related to sequences obtained from carnivores (97.2% identical), and the sequence from the boa constrictor was most closely related to the rodent cluster (97.4% identical). These data are consistent with recent work identifying prey-predator transmission cycles in Hepatozoon spp. and suggest this pattern may be more common than previously recognized.

Intraerythrocytic parasites (Apicomplexa: Sarcocystidae) of the South American mouse opossum (Thylamys elegans) from Chile, South America, and of the yellow-bellied glider (Petaurus australis) from Australia were found to be monophyletic using SSU rDNA and partial LSU rDNA sequences. Phylogenetic reconstruction placed both species within the family Sarcocystidae. These intraerythrocytic parasites of marsupials represent an as yet unnamed genus predicted to have bisporocystic oocysts and tetrazoic sporocysts, which is a characteristic feature of all members of the family Sarcocystidae. These results show that erythrocytic parasites share a common ancestor and suggest co-evolution with their vertebrate host.

This is a case report of natural infection with Besnoitia darlingi in a Virginia opossum (Didelphis virginiana) in Louisiana. Clinical pathologic data included a severe nonregenerative anemia, inflammatory leukogram, increased hepatocellular leakage enzymes, renal azotemia, hyperkalemia, hypoglycemia, hypoalbuminemia, and proteinuria. Tissue cysts containing bradyzoites were found in the majority of organs, especially the skin, mucous membranes, kidneys, adrenals, lungs, and heart. Images of the bradyzoites obtained by transmission electron microscopy were consistent with the previously described ultrastructure of Besnoitia darlingi. This opossum also suffered from an open phalangeal fracture and concurrent gastrointestinal parasites. Histopathologic findings included a glomerulonephritis and hepatic necrosis.

The blood of 21 adult South American mouse opossums (Thylamys elegans) captured from April through August of 2005 in central Chile was examined for parasites. Light microscopic analysis of blood smears initially suggested that a highly pleomorphic Hepatozoon species typical of American opossums was infecting erythrocytes. Unexpectedly, amplification by PCR and sequencing of a DNA fragment of the small subunit rDNA combined with phylogenetic analyses indicated that the parasite is not a member of the suborder Adeleorina, which includes the Haemogregarina and Hepatozoon species, but that it is a clearly distinct member of the suborder Eimeriorina, which includes the cyst-forming family Sarcocystidae. Therefore, a reclassification of this unusual intraerythrocytic apicomplexan will require additional life cycle, microscopic, and molecular analyses.

Tachyzoites of Besnoitia darlingi Brumpt, 1913 were redescribed based on new materials isolated from Virginia opossums (Didelphis virginiana, Kerr) from Michigan, U.S.A. Tachyzoites of the MIBD1 strain were propagated in bovine turbinate cell culture for more than two years. A comparison with previously described tachyzoites of the B. darlingi OP1 strain from Mississippi, USA revealed some morphological differences despite the remarkable genetic homogeneity between the two B. darlingi strains. MIBD1 tachyzoites were distinguished from OP1 tachyzoites by having more rhoptries, and fewer and haphazardly distributed micronemes at the conoidal end. This morphological heterogeneity between tachyzoites of the two strains suggests the role of geographical isolation in the Michigan strain. New morphological features of B. darlingi tachyzoites were described.

Besnoitia tissue cysts were found in five naturally-infected adult opossums (Didelphis virginiana) from Michigan. Details of the microscopy, histopathology, ultra-structure, and genetic features of the cysts were studied to identify their species-specific traits. The materials were differentiated phenotypically from cysts of other Besnoitia spp. by difference in size, pattern of tissue distribution, morphology of pellicle and nucleus, number of micronemes and rhoptries, amount of lipids and amylopectin, and presence of enigmatic bodies. Morphometric variations identified the tissue cysts and the pathologic changes in opossums host to be due to B. darlingi. The data were proved by phylogenetic analysis based on DNA sequences of the first internal transcribed spacer of nuclear rDNA. Cluster analysis showed that B. darlingi was distinct from all other Besnoitia spp. as two distinct phylogenetic clades: I- included Besnoitia spp. described from opossum (B. darlingi), sheep (B. jellisoni), rodent (B. akadoni) and rabbit (B. oryctofelisi) and clade II- encompassed parasites described from cattle (B. besnoiti), equids (B. bennetti) and reindeer (B. tarandi). The genetic attributed particular to the genus Besnoitia complemented the morphologica features and lead to accurate delimitation of Besnoitia species.

The common brushtail possum (Trichosurus vulpecula) has well adapted to increasing urbanization, resulting in greater interaction with humans and their domestic pets. Wildlife species in urban areas face a higher risk of exposure to zoonotic pathogens and may be affected by parasites hosted by cats (Toxoplasma gondii) or dogs (Neospora caninum), yet it is unknown to what extent urban T. vulpecula are exposed to these parasites. Antibodies to T. gondii and N. caninum were assayed in sera of 142 adult possums from the city of Sydney, Australia. Using the modified agglutination test, antibodies to T. gondii were found in 9 (6.3%) of the 142 animals in titers of 1:25 (4), 1:50 (1), 1:100 (1), 1:800 (1), 1:3,200 (1), 1:6,400 (1), and 1:12,800 (1). Of some T. vulpecula multiple sera samples within a 2-yr frame could be collected, but seropositive animals in general were not recaptured after initial seroconversion. One possum had a high T. gondii titer on 2 consecutive bleedings, 14 mo apart, and seropositive possums appeared normal when captured. Sex seemed not to have an affect on antibody prevalence, but age and location may play a role. Antibodies to N. caninum were not detected in 1:25 dilution of sera in the N. caninum agglutination test, indicating that T. vulpecula may not have been exposed to this parasite. This is the first serological survey for T. gondii and N. caninum infections in urban T. vulpecula.

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.

The Sarcocystidae comprise a diverse, monophyletic apicomplexan parasite family, most of whose members form intracellular cysts in their intermediate hosts. The extent of pathology associated with such cyst formation can range widely. We currently lack experimental animal models for many of these infections. Here we explored dexamethasone treatment as a means to render outbred mice susceptible to Besnoitia darlingi infection and demonstrated that this approach allows viable parasites to be subsequently isolated from these mice and maintained in tissue culture. Besnoitia bradyzoites recovered from crushed cysts derived from naturally infected opossums (Didelphis virginiana) replicated and reproduced the development of besnoitiosis in mice treated with dexamethasone (0.5 mg/ml drinking water) daily for 12 days post infection (DPI). Isolates recovered from the peritoneal exudates of these mice were viable and were maintained in long-term tissue cultures. In contrast, control mice given saline without dexamethasone and challenged with similar bradyzoites remained clinically normal for up to 70 DPI. An additional group of mice challenged with the same inoculum of bradyzoites and given dexamethasone at the same concentration and treated with sulfadiazine (1 mg/ml drinking water) daily for 12 DPI also remained normal for up to 70 DPI. Severe disease developed more rapidly in dexamethasone-treated mice inoculated with culture-derived B. darlingi tachyzoites than in those inoculated with cyst-derived bradyzoites. B. darlingi tachyzoite-infected, untreated control mice developed signs of illness at 18 DPI. In contrast, mice treated with sulfadiazine showed no clinical signs up to 50 DPI. Although dexamethasone treatment was required to establish B. darlingi infection in outbred mice inoculated with opossum-derived B. darlingi bradyzoites, no such treatment was required for mice inoculated with culture-derived B. darlingi tachyzoites. Finally, sulfadiazine was highly effective in protecting mice from infection with the tachyzoite stage of B. darlingi.

Opossums (Didelphis virginiana) are exposed to a wide range of coccidia through feeding on a variety of foods, including, but not limited to, carrion, insects, and nestling birds. Abundant D. virginiana populations in urban and suburban areas can be important reservoirs of parasitic infection because of their profuse and prolonged excretion of the sporocysts of several species of Sarcocystis, their omnivorous diet, and their relatively long life span. This report describes 2 adult female opossums found to be simultaneously infected with the tissue cysts of Besnoitia darlingi, sarcocysts of Sarcocystis inghami, as well as with the intestinal sporocysts of S. neurona. Cysts typical of B. darlingi based on gross, histological, and ultrastructural characteristics were disseminated throughout the visceral organs, musculature, ears, and skin. The S. neurona and B. darlingi infections were confirmed by comparative sequence analysis of polymerase chain reaction-amplified diagnostic genetic loci. Sarcocysts of S. inghami are also described. Such examples of multiple parasitic infections show that concurrent infections occur naturally. The propensity for species to coexist should be considered in the differential diagnosis of tissue cyst-forming coccidian protozoa and may have important epidemiological and evolutionary implications.

Specimens of Virginia opossums (Didelphis virginiana) in Michigan were examined over 1 year to document the presence of Besnoitia darlingi cysts. Cyst morphology, prevalence, seasonal variation, and tissue sites of isolation were studied. Histology and ultrastructural features of the detected cysts and bradyzoites were consistent with B. darlingi. In the opossums, B. darlingi had intracellular tissue cysts. Tissue cysts had a mean diameter of 560 microm and were separated from the host tissue by a thick (5-20 microm) cyst wall. Overall prevalence of B. darlingi cysts in opossums was 10.9% (15/137). Variations in the prevalence were detected during spring (3/17; 17.6%), summer (10/34; 29.4%), and fall (2/60; 3.3%). No cysts were detected in the specimens examined during winter (0/26; 0%). Numerous B. darlingi cysts were detected in ears, conjunctiva, tongue, abdominal muscles, diaphragm, stomach, heart, liver, kidney, lung, and spleen. Cysts were detected mainly in adult female opossums that were debilitated. Ear was the most frequent organ from which the cysts were reported (10/15; 66.7%) when compared individually with other body tissues (P<0.05).

Antibodies to Neospora caninum and Toxoplasma gondii were assayed in sera of 396 opossums (Didelphis marsupialis) from the city of São Paulo, Brazil. Antibodies to N. caninum were assayed using the indirect immunofluorescent antibody test (IFAT). Antibodies (IFAT, approximately 1:25) to N. caninum were found in 84 opossums (D. marsupialis) in titers of 1:25 in 46, 1:50 in 20, 1:100 in 17, and 1:400 in 1. Antibodies to T. gondii were assayed with the modified agglutination test (MAT) and the IFAT. Antibodies to T. gondii (MAT, approximately 1:25) were found in 82 (20.4%) of the 396 opossums, in titers of 1:25 in 24, 1:50 in 26, 1:100 in 18, 1:200 in 13, and 1:800 in 1. The IFAT antibodies to T. gondii were found in 148 of 396 opossums, in titers of 1:16 in 41, 1:32 in 23, 1:64 in 13, 1:128 in 6, 1:256 in 20, 1:512 in 17, 1:1,024 in 10, 1:2,048 in 10, 1:4,096 in 7, and 1:8,192 in 1. This is the first report of N. caninum and T. gondii infections in D. marsupialis.

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.

Faecal samples collected from 300 Bolivian marsupials (Didelphimorphia: Didelphidae) between 1984 and 1993 were examined for coccidian parasites. Sporulated oocysts were present in the faeces of 50 (17%) marsupials representing 11 genera and 22 species. Three new species of Eimeria are described and named from six host species. One species occurred in Marmosops dorothea, Monodelphis domestica and Thylamys venustus, another in Micoureus constantiae constantiae and Micoureus constantiae budini and a third in Marmosops dorothea. A discriminant analysis performed on five quantitative oocyst measurements revealed similarities between the first and third Eimeria species because of similar sizes and shapes of the oocysts, whereas the second Eimeria species was structurally discrete. The Eimeria that infects multiple hosts may be a common widespread species. Future surveys are advised for a thorough assessment of the coccidian biodiversity within Bolivian marsupials.