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A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study.

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought.

Milk provides mammalian neonates with nutritional support and passive immunity. This is particularly true in marsupials where young are born highly altricial and lacking many components of a fully functional adaptive immune system. Here we investigated the T cell populations in the mammaries of a lactating marsupial, the gray short-tailed opossum Monodelphis domestica. Immunohistochemistry confirmed the presence of T cells within the opossum mammaries throughout lactation. Results of quantifying transcript abundance for lymphocyte markers are consistent with γδ T cells being the most common T cell type within lactating mammaries. Numbers of γδ T cells appear to peak early during the first postnatal week, and then decline throughout lactation until weaning. In contrast, numbers of αβ T cells and γμ T cells appear to be low to non-existent in the lactating mammaries. The results support an ancient and conserved role of immune cells in the evolution and function of mammalian mammary tissue.

The early loss of vision results in a reorganization of visual cortex, which becomes commandeered by the somatosensory and auditory systems. Recent studies in the short-tailed opossum found that the connections and response properties of neurons in somatosensory cortex of blind animals are also altered. While studies in humans and other mammals show that early vision loss leads to heightened abilities on discrimination tasks involving the spared senses, if and how this superior discrimination leads to adaptive sensorimotor behavior has yet to be determined. Moreover, little is known about the extent to which blind animals rely on the spared senses. Here, we tested opossums on a sensorimotor task involving somatosensation. We found that early blind animals had increased limb placement accuracy compared to sighted controls, while showing similarities in crossing strategy. Whisker trimming nullified this advantage, resulting in decreases in limb placement accuracy and more variable forelimb movements in both groups. However, the increased reliance on tactile inputs in early blind animals led to greater deficits in limb placement and behavioral flexibility when the whiskers were trimmed. This data demonstrates that incoming sensory input from the whiskers is processed differently in the brains of early blind compared to sighted opossums, likely due, in part, to the dramatic changes to visual and somatosensory cortex. These results also provide direct evidence for the prevailing theory that the whiskers are critical for skilled forelimb control in whisking mammals, and highlight the importance of studying both performance and strategy when assessing differences in behavior.

B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. The marsupial immunoglobulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confirm greater diversity provided by the L chain V. This study is the first to report on using single cell analysis to investigate Ig repertoires in a marsupial and confirms a number of prior hypothesis, as well as revealing some surprises.

Summary Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

From 1957 to 1962, thalidomide caused birth defects in >10,000 children. While the drug was pulled from the market, thalidomide is currently prescribed to treat conditions including leprosy. As a result, a new generation of babies with thalidomide defects is being born in the developing world. This represents a serious problem, as the mechanisms by which thalidomide disrupts development remain unresolved. This lack of resolution is due, in part, to the absence of an appropriate mammalian model for thalidomide teratogenesis. We test the hypothesis that opossum (Monodelphis domestica) is well suited to model human thalidomide defects. Results suggest that opossum embryos exposed to thalidomide display a range of phenotypes (e.g., heart, craniofacial, limb defects) and penetrance similar to humans. Furthermore, all opossums with thalidomide defects exhibit vascular disruptions. Results therefore support the hypotheses that opossums make a good mammalian model for thalidomide teratogenesis, and that thalidomide can severely disrupt angiogenesis in mammals.

Marsupial reproduction differs significantly in anatomy and physiology from that of placental mammals. The gastrointestinal and urogenital tracts empty into a cloaca. Reproductive parameters include gestation and pouch time, as the fetus develops outside of the uterus. Captive marsupials discussed include sugar gliders), short-tailed opossums, Virginia opossums, and Tammar and Bennett's wallabies. Common disease conditions include joey eviction, pouch infections, mastitis, metritis, prostatitis, penile necrosis, and neoplasia. Common surgeries include penile amputation, castration, and ovario-vaginal-hysterectomy or ovario-hysterectomy. Therapeutics used for these conditions are presented.

Odontogenesis is guided by a complex signaling cascade in which several molecules, including FGF2-4, ensure all dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is an opossum with the closest dentition to humans, and the main odontogenesis stages occur when the newborns are in the pouch. In this study, D. albiventris postnatals were used to characterize the main stages of their molars development; and also to establish FGF2, FGF3 and FGF4 expression pattern. D. albiventris postnatals were processed for histological and indirect immunoperoxidase analysis of the tooth germs. Our results revealed similar dental structures between D. albiventris and mice. However, FGF2, FGF3 and FGF4 expression patterns were observed in a larger number of dental structures, suggesting broader functions for these molecules in this opossum species. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth in humans. This study may also contribute to the implementation of D. albiventris as model for Developmental Biology studies.

South American opossums of the order Didelphimorphia are considered sentinels for zoonotic infections and environmental diseases, such as for Chagas disease caused by Trypanosoma cruzi. Nevertheless, there is a paucity of data regarding protozoan diseases such Toxoplasma gondii and Neospora caninum in Neotropical marsupials; despite these pathogens have been considered threats to some marsupial species. The aim of this study was to determine whether Didelphis albiventris and Philander frenatus opossums from southern Brazil had been previously exposed to T. cruzi, T. gondii or N. caninum. Opossum samples were obtained by live-trapping of free-ranging animals and collection at wildlife rehabilitation centers in Rio Grande do Sul (RS) state, Brazil. The detection of anti-T. cruzi and anti-T. gondii antibodies was performed by indirect hemagglutination and anti-N. caninum antibodies by competitive enzyme-linked immunosorbent assay. In total, samples were collected from 76 marsupials from nine municipalities in RS state, including 69 D. albiventris (white-eared opossum), and seven were P. frenatus (southern four-eyed opossum). For T. cruzi, 11% were seropositive, and for T. gondii 26% were seropositive. None of the marsupials sampled here were seropositive for N. caninum. Risk factor analysis showed that free-living animals were about five-fold more likely to be infected by T. gondii than were rescued animals. Our study showed the exposure of Neotropical marsupials (D. albiventris and P. frenatus) to protozoan pathogens T. cruzi and T. gondii, while no evidence of N. caninum exposure was found. The set of results presented here may have an effect on ecology and conservation of the studied species and may also indicate possible sentinels of these pathogen circulation.

Expressed coding sequences for interleukin-6 (IL-6) and interleukin-6 receptor α (IL-6R) were examined in five marsupial species. Full length expressed coding sequences for IL-6 and IL-6R were identified and characterized in the gray short-tailed opossum (Monodelphis domestica). For IL-6, ∼225 bp fragments of the mRNA sequence were identified in the red-tailed phascogale (Phascogale calura), kultarr (Antechinomys laniger), and stripe-faced dunnart (Sminthopsis macroura), while ∼563 bp fragments of mRNA encoding IL-6R were identified in the red-tailed phascogale, kultarr, stripe-face dunnart and fat-tailed dunnart (Sminthopsis crassicaudata). Relative expression levels of IL-6 and IL-6R were examined in the heart, muscle, lung, liver, spleen and kidney of adult red-tailed phascogales, and IL-6 gene expression was found to be significantly higher in the lung and spleen than the other tissues examined, while the expression of IL-6R was significantly higher in the liver, lung and spleen. These results now serve as a reference point for examining the role and levels of IL-6 and IL-6R in the health and disease of these marsupial species. The pro-tumorigenic nature of IL-6 is of particular interest, and the identification of these IL-6 and IL-6R coding sequences provides a platform for further work to evaluate the potential role of IL-6 in marsupial cancers.

The Water Opossum, Chironectes minimus (Didelphimorphia, Didelphidae), is the only semi-aquatic marsupial and can be easily identified for its uniquely patterned water resistant pelage and webbed hind-feet. The species is distributed along the Neotropical region (from Southern Mexico to Northwestern Argentina) and is currently divided on four subspecies (C.m. argyrodytes, C. m. panamensis, C. m. minimus, C. m. paraguensis), based on its geographical distribution. We evaluated the geographic variation in C. minimus, using traditional and geometric morphometrics techniques to capture the size and shape of the skull to identify differences between the populations that could relate to existing subpsecies or populations that and would merit particular taxonomic attention. We took 13 skull measurements, and digitized 37, 18, and 23 2D landmarks on the dorsal and lateral view of the cranium and on the lateral view of the mandible, respectively. The sample consisted on 135 individuals from 66 localities. Analyses of both datasets showed that the Southern South America and Northern Central America animals differ from the other populations. Geometric morphometrics shows on the three views that the Amazon animals differ from the others probably due to existing geographical barriers. These morphological differences among populations of the Water Opossum support the need for a proper phylogenetic assessment of these populations, in order to clarify their taxonomic status.

We aimed to evaluate the role of spatial units with different shapes and sizes on road-kill modeling for small vertebrate species. We used the road-kill records of two reptiles, water snake ( Helicops infrataeniatus ) and D’Orbigny's slider turtle ( Trachemys dorbigni ), and three mammals, white-eared opossum ( Didelphis albiventris ), coypu ( Myocastor coypus ) and Molina's Hog-nosed skunk ( Conepatus chinga ). Hierarchical partitioning was used to evaluate the independent influence of different land-use classes on road-kill by varying the shape and size of the spatial units. Variables that most explained road-kill were consistent over the different spatial unit types. The standard size seemed to be a reasonable solution for these species. Prior analysis with several sizes and shapes is needed to identify the appropriate spatial unit to model road-kill occurrence for larger vertebrates with different history traits.

Trypanosoma cruzi is the etiologic agent of American trypanosomiasis and can infect humans and different species of domestic and wild animals. The marsupials are important wild reservoirs of T. cruzi, aiding in the maintenance of this agent in sylvatic and peri-domestic environments. The objective of this study was to report the parasitological and clinicopathological findings of a natural infection by T. cruzi in one specimen of Philander opossum that originated from the Brazilian Amazon. The animal was captured in a forest fragment near a rural community with reports of human Chagas disease. T. cruzi infection was diagnosed by blood smear examinations, blood culture, scent glands secretion culture, histopathological examination, and nested-PCR. Positive samples were subjected to PCR to characterize the discrete typing units (DTUs) of T. cruzi. Characteristic trypomastigotes of T. cruzi were observed in the blood smear, and spheromastigotes, epimastigotes, and trypomastigotes were visualized in the cultures. Non-suppurative myocarditis associated with amastigote clusters was the principal histopathological finding. DNA from T. cruzi was detected in samples of blood, blood cultures, scent glands secretion cultures, cardiac muscles, and the spleen. The TcI and the TcII/V/VI group DTUs were detected in blood culture and scent glands secretion cultures. Infection by T. cruzi can cause myocarditis in P. opossum and DTUs TcI and TcII/V/VI group mixed infection can be detected in the acute phase. P. opossum can be a source of infection for triatomine vectors and has the potential source for direct transmission of T. cruzi by secretions from the scent glands. These data are important to improve the understanding of the complex enzootic transmission cycle of T. cruzi in the Brazilian Amazon.

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.

Made available in DSpace on 2020-12-12T02:11:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Universidade Estadual Paulista Worldwide, Bartonella species are known to infect a wide range of mammalian and arthropod hosts, including humans. The current study aimed to investigate the prevalence of Bartonella spp. in synanthropic mammals captured in peri-urban areas from Central-Western and Southern Brazil and their ectoparasites. For this aim, 160 mammals belonging to four species, and 218 associated arthropods were sampled. DNA was extracted and subjected to different Bartonella screening assays. Additionally, blood samples from 48 small rodents were submitted to liquid BAPGM culture followed by qPCR assay and solid culture. Two out of 55 Rattus captured in Santa Catarina state were PCR-positive for Bartonella when targeting the nuoG, 16S, and ITS loci. Sequences showed high homology with Bartonella coopersplainsensis. Conversely, all 48 small rodents, 14 capybaras and 43 opossum DNA samples from animals trapped in Mato Grosso do Sul were Bartonella negative in the HRM real time PCR assays targeting the ITS locus and gltA gene. Additionally, all mammal-associated ectoparasites showed negativity results based on HRM real time PCR assays. The present study showed, for the first time, the occurrence of B. coopersplainsensis in Brazil, shedding some light on the distribution of rats-related Bartonella in South America. In addition, the majority of rodents and marsupials were negative for Bartonella spp. Since B. coopersplainsensis reservoirs - Rattus spp. - are widely dispersed around the globe, their zoonotic potential should be further investigated. Graduate Program of Agricultural and Livestock Microbiology Faculty of Agrarian and Veterinary Sciences (FCAV/UNESP) Department of Veterinary Pathology Faculty of Agrarian and Veterinary Sciences (FCAV/UNESP) Koret School of Veterinary Medicine The Robert H. Smith Faculty of Agriculture Food and Environment. The Hebrew University of Jerusalem Dom Bosco Catholic University Department of Veterinary Medicine University of Contestado Federal University of Mato Grosso do Sul Graduate Program of Agricultural and Livestock Microbiology Faculty of Agrarian and Veterinary Sciences (FCAV/UNESP) Department of Veterinary Pathology Faculty of Agrarian and Veterinary Sciences (FCAV/UNESP)

Spirura genus Blanchard, 1849 comprise of nematode parasites that infect primate and marsupial species. Although several taxonomical studies have shown that the infection by this species occurs primarily in the esophagus of primates, evidence for the occurrence of these parasites in other hosts (marsupials, rodents and bats) has become the subject of investigation by several groups. In this work, we describe the presence of Spirura guianensis Ortlepp, 1924 in the marsupial Gracilinanus agilis (Marsupialia: Didelphidae) found in the Pantanal of Mato Grosso do Sul state of Brazil. Structural characteristics of this nematode were identified using light microscopy (bright field and fluorescence stereomicroscopy) and scanning electron microscopy (SEM) approaches. Details of the surface topography such as cephalic projections, ventral boss, details of the caudal papillae and cuticular ornamentations were shown, providing taxonomic characteristics that may help in the establishment of diagnostic protocols. In addition, the presence of this species in a new host and new geographical area of Brazil provide grounds for a revision on the distribution of S. guianensis in South America.

The Virginia opossum (Didelphis virginiana) and the striped skunk (Mephitis mephitis) are often kept as pets, although they are both wild animals. The opossum and striped skunk are usually docile and sociable when raised from infancy. Many states and municipalities restrict ownership, and veterinarians should familiarize themselves with local laws and regulations pertaining to these animals. The biology, husbandry, diet, and general disease information is available for both species. Vaccination recommendations are included for skunks.

Marsupials comprise an interesting group of mammals, which are increasingly being kept as pets. Few actual feeding trials have been published, although many anecdotal diets have years of usage with good success. Marsupials have dental and digestive tract adaptations that allow them to use specific niches in their environments. Knowing the diet in the wild is instrumental in designing diets used in captivity.

The gray short-tailed opossum (Monodelphis domestica) is a nocturnal South American marsupial that has been gaining popularity as a laboratory animal. However, compared to traditional laboratory animals like rats, very little is known about its behavior, either in the wild or in a laboratory setting. Here we investigated the photic preference of the short-tailed opossum. Opossums were placed in a circular testing arena and allowed to move freely between dark (0 lux) and light (~1.4, 40, or 400 lux) sides of the arena. In each of these conditions opossums spent significantly more time in the dark than in the illuminated side and a greater proportion of time in the dark than would be expected by chance. In the high-contrast (~400 lux) illumination condition, the mean bout length (i.e., duration of one trip on the light or dark side) was significantly longer on the dark side than on the light side. When we examined the number of bouts greater than 30 and 60 sec in duration, we found a significant difference between the light and dark sides in all light contrast conditions. These data indicate that the short-tailed opossum prefers the dark to the light, and can also detect very slight differences in light intensity. We conclude that although rats and opossums share many similar characteristics, including ecological niche, their divergent evolutionary heritage results in vastly different behavioral capabilities. Only by observing the behavioral capabilities and preferences of opossums will we be able to manipulate the experimental environment to best elicit and elucidate their behavior and alterations in behavior that can arise from experimental manipulations.

Lyn is a tyrosine kinase molecule required for modulation of signalling cascades in cell populations including B lymphocytes of the mammalian immune system. We have characterised the coding domain of the marsupial lyn gene of two macropod marsupials; the Tammar Wallaby (Macropus eugenii) and the Bridled Nailtail Wallaby (Onychogalea fraenata) and show the co-expression of two Lyn isoforms in cells and tissues of these and three other marsupials (Brush-tail Possum, Trichosurus vulpecula; American Grey Short-tailed Opossum, Monodelphis domestica and Red-tailed Phascogale, Phascogale calura). The predicted Lyn proteins (LynA and LynB) were highly conserved across vertebrate species, with amino acid identities of 94% with their human orthologues and conservation of key tyrosine kinase motifs that suggests that marsupial Lyn most likely functions in cell signalling. Comparison of our cDNA data to annotations for Lyn transcripts (available through the Ensembl Genome Browser) for the Tammar Wallaby confirm splice sites for a number of exons in the wallaby transcript that are missing from the current annotation. This is the first report of the expression of kinase signalling molecules that influence immunity in metatherian mammals and provides key information to support ongoing studies of immune regulation in marsupials.

Internal parasitic infections are common in the Virginia opossum ( Didelphis virginiana ). More than 50 different species of nematodes have been identified from the genus Didelphis . Of them, 3 can cause significant illness to native opossums in Southern California, 2 gastrointestinal nematodes and 1 pulmonary nematode. This article provides an overview of the life cycles, methods for identification, pathologic findings, diagnostic tests, clinical signs, and options for treating these common nematode species.

Colour vision in marsupials is a controversial issue, especially among the genus Didelphis (Didelphidae, Didelphimorphia). While behavioural tests have diagnosed these animals as trichromats and electrophysiological studies have diagnosed them as monochromats, recent molecular genetics studies provide evidence for dichromatic colour vision, having found two classes of cone opsins in a species of this genus. This study examines the colour perception of a male and female white-eared opossum, Didelphis albiventris, through a series of tasks involving a behavioural paradigm of discrimination learning. Both opossums succeeded in discriminating pairs of stimuli consisting of Munsell colour cards presented in random brightness values that are assumed to be easily discriminated by dichromats and trichromats (e.g. blues versus oranges). However, both subjects failed to discriminate between colours that are expected to be easily discriminated only by trichromats (e.g. greens versus oranges). The opossums were also unsuccessful in distinguishing a colour against itself (e.g. oranges versus oranges), demonstrating that discrimination was based only on visual cues. These results are consistent with recent predictions based on molecular genetics suggesting that the genus Didelphis is routinely a dichromat.

Changes in gene expression have been measured 24h after injury to mammalian spinal cords that can and cannot regenerate. In opossums there is a critical period of development when regeneration stops being possible: at 9 days postnatal cervical spinal cords regenerate, at 12 days they do not. By the use of marsupial cDNA microarrays, we detected 158 genes that respond differentially to injury at the two ages critical for regeneration. For selected candidates additional measurements were made by real-time PCR and sites of their expression were shown by immunostaining. Candidate genes have been classified so as to select those that promote or prevent regeneration. Up-regulated by injury at 8 days and/or down-regulated by injury at 13 days were genes known to promote growth, such as Mitogen-activated protein kinase kinase 1 or transcription factor TCF7L2. By contrast, at 13 days, up-regulation occurred of inhibitory molecules, including annexins, ephrins, and genes related to apoptosis and neurodegenerative diseases. Certain genes such as calmodulin 1 and NOGO, changed expression similarly in animals that could and could not regenerate without any additional changes in response to injury. These findings confirmed and extended changes of gene expression found in earlier screens on 9 and 12 ay preparations without lesions and provide a comprehensive list of genes that serve as a basis for testing how identified molecules, singly or in combination, promote and prevent central nervous system regeneration.

Sarcocystis neurona is a significant cause of neurological disease in horses and other animals, including the threatened Southern sea otter (Enhydra lutris nereis). Opossums (Didelphis virginiana), the only known definitive hosts for S. neurona in North America, are an introduced species in California. S. neurona DNA isolated from sporocysts and/or infected tissues of 10 opossums, 6 horses, 1 cat, 23 Southern sea otters, and 1 harbor porpoise (Phocoena phocoena) with natural infections was analyzed based on 15 genetic markers, including the first internal transcribed spacer (ITS-1) region; the 25/396 marker; S. neurona surface antigen genes (snSAGs) 2, 3, and 4; and 10 different microsatellites. Based on phylogenetic analysis, most of the S. neurona strains segregated into three genetically distinct groups. Additionally, fifteen S. neurona samples from opossums and several intermediate hosts, including sea otters and horses, were found to be genetically identical across all 15 genetic markers, indicating that fatal encephalitis in Southern sea otters and equine protozoal myeloencephalitis (EPM) in horses is strongly linked to S. neurona sporocysts shed by opossums.

Sarcocystis neurona, a protozoal parasite shed by opossums (Didelphis virginiana), has been shown to cause significant morbidity and mortality in horses, sea otters, and other marine mammals. Over the course of 3 years (fall 2005-summer 2008), opossums from central California were tested for infection with S. neurona. Of 288 opossums sampled, 17 (5.9%) were infected with S. neurona based on the molecular characterization of sporocysts from intestinal scrapings or feces. Risk factors evaluated for association with S. neurona infection in opossums included: age, sex, location, season, presence of pouch young in females, concomitant infection, and sampling method (live-trapped or traffic-killed). Multivariate logistic regression analysis identified that opossums in the Central Valley were 9 times more likely to be infected than those near the coast (p=0.009). Similarly, opossum infection was 5 times more likely to be detected during the reproductive season (March-July; p=0.013). This first investigation of S. neurona infection prevalence and associated risk factors in opossums in the western United States can be used to develop management strategies aimed at reducing the incidence of S. neurona infections in susceptible hosts, including horses and threatened California sea otters (Enhydra lutris neries).

Using Sulforhodamine-101 (SR101) labeling and calcium imaging on in vitro preparations, we investigated the development of spontaneous activity in the spinal enlargements of a marsupial born more immature than eutherian mammals, the opossum Monodelphis domestica. Following the retrograde transport of Calcium Green dye from the limb nerves, we observed the occurrence of spontaneous calcium waves activating the motor columns of the cervical enlargement of opossums aged from P3 to P15 (day of birth: P0) and of the lumbar enlargement from at least P6 to P12. In other preparations, SR101 was added to the bath to identify the active cells. In P1 opossums, only a few SR101-labeled cells were observed in the cervical enlargement and none in the lumbar enlargement. At P5, their number increased cervically and they appeared in the lumbar enlargement. Motoneurons were the major cell type labeled by SR101 but dye leakage made their quantification inaccurate. SR101-labeled cells also occurred elsewhere in the ventral and dorsal grey. Their number increased until P12–14 in both enlargements and then decreased to disappear by P21, the last age examined. Thus in contrast to eutherian mammals, in which spontaneous activity is mostly prenatal, spontaneous activity occurs predominantly postnatally in opossums. It increases at the time when connections from the brain begin to impinge on spinal neurons and when the limbs, especially the hindlimbs, start moving and then decreases as the systems mature.

In eutherian mammals, CD8 is a key receptor of cytotoxic T cells and plays a pivotal role in the recognition and elimination of infected host cells by cell-mediated cytotoxicity. Here, we report the molecular cloning and expression analysis of CD8alpha and CD8beta cDNAs in two marsupial species, the gray short-tailed opossum and the tammar wallaby. The opossum and tammar CD8 sequences share a high degree of amino acid identity of 63% (CD8alpha) and 57% (CD8beta) to each other as well as 36-45% (CD8alpha) and 38-41% (CD8beta) with their eutherian counterparts. In addition, many of the signature features of eutherian CD8alpha and CD8beta are preserved in both marsupials including the two invariant cysteines that form the intra-chain disulphide bond in the extracellular IgSfV domain and the two hinge region cysteines involved in dimerisation between the two subunits. The p56(lck) binding motif in the cytoplasmic tail of the CD8alpha subunit is also conserved. Interestingly, the opossum CD8alpha and the tammar CD8beta sequences have a truncated cytoplasmic tail. RT-PCR analysis of CD8alpha and CD8beta transcripts in the tissues of the adult opossum and tammar showed broad tissue expression with a high level of expression observed in the lymphoid tissues of both marsupials. Furthermore, RT-PCR analysis of CD8alpha and CD8beta transcripts in the immune tissues of tammar young over the first 120 days of pouch life revealed a pattern of expression analogous to the maturation of the lymphoid tissues. This is the first report confirming the presence of CD8 in the tissues of a marsupial and will provide the tools to further analyse T cell subsets in this unique group of mammals.

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.

Summary. Background: Previous reports have noted that factor (F) XI and FXII and prekallikrein (the contact phase proteases) are absent in fish. Objectives: A broad survey of recently completed genomes was undertaken to find where during the course of vertebrate evolution these coagulation factors appeared. Methods: BLAST searches were conducted for the various factors on genomes of lamprey, puffer fish, zebra fish, frog, chicken, platypus, and opossum. Results: It was confirmed that FXII is absent from fish; it is present in frog, platypus, and opossum, but is absent in chicken, an apparent example of gene loss. A single gene corresponding to the evolutionary predecessor of FXI and prekallikrein occurs in frog, chicken, and platypus. The opossum (a marsupial) has both prekallikrein and FXI, completing the full complement of these genes that occurs in eutherian mammals. Conclusions: The step-by-step accrual of genes for these factors by a series of timely gene duplications has been confirmed by phylogenetic analysis and other considerations.

We investigated nuclear divisions of the thalamus in the gray short-tailed opossum (Monodelphis domestica) to gain detailed information for further developmental and comparative studies. Nissl and myelin staining, histochemistry for acetylcholinesterase and immunohistochemistry for calretinin and parvalbumin were performed on parallel series of sections. Many features of the Monodelphis opossum thalamus resemble those in Didelphis and small eutherians showing no particular sensory specializations, particularly in small murid rodents. However, several features of thalamic organization in Monodelphis were distinct from those in rodents. In the opossum the anterior and midline nuclear groups are more clearly separated from adjacent structures than in eutherians. The dorsal lateral geniculate nucleus (LGNd) starts more rostrally and occupies a large part of the lateral wall of the thalamus. As in other marsupials, two cytoarchitectonically different parts, alpha and beta are discernible in the LGNd of the opossum. Each of them may be subdivided into two additional bands in acetylcholinesterase staining, while in murid rodents the LGNd consists of a homogeneous mass of cells. Therefore, differentiation of the LGNd of the Monodelphis opossum is more advanced than in murid rodents. The medial geniculate body consists of three nuclei (medial, dorsal and ventral) that are cytoarchitectonically distinct and stain differentially for parvalbumin. The relatively large size of the MG and LGNd points to specialization of the visual and auditory systems in the Monodelphis opossum. In contrast to rodents, the lateral dorsal and lateral posterior nuclei in the opossum are poorly differentiated cytoarchitectonically.

We tested the hypothesis that brown-headed cowbirds ( Molothrus ater ) harbor Sarcocystis neurona , the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona . Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4 ± 0.7 μm by 7.6 ± 0.4 μm ( n = 25) and 12.6 ± 0.6 μm by 8.0 ± 0 μm ( n = 25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNγ knockout mice indistinguishable from that of the positive control inoculated with S. neurona . This, together with the knowledge that S. falcatula does not cause lesions in IFNγ knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona . Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona ; thin-walled sarcocysts typed as S. neurona . In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5 g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona -specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.

Fatty acids are potential trophic markers to trace feeding relationships in aquatic ecosystems primarily because lipid reserves of organisms broadly reflect dietary sources of lipids and can therefore provide information on the availability of key fatty acids in the food web. However, the use of fatty acids for such applications may be constrained by the degree to which the fatty acid composition of organisms is obscured by factors other than straightforward uptake from the diet. Thus, we studied the effect of long-term fasting, under controlled laboratory conditions, on the lipid content and fatty acid composition of field-caught Mysis relicta. Periods of 3 to 6 weeks are required to induce clear effects of fasting in M. relicta. Relative proportions of docosahexaenoic acid (DHA; 22:6n-3) and arachidonic acid (ARA; 20:4n-6) increased with decreasing lipid contents during fasting. DHA proportions in the total fatty acid composition of fasting M. relicta were significantly higher in comparison to field-caught animals. M. relicta with high proportions of DHA (> 25%) and with low lipid contents (< 14% of dry weight) can be clearly identified as fasting animals. Such thresholds will help to improve the validity of fatty acids in trophic studies.

The purpose of this study was to characterize cytogenesis and apoptosis in the developing retina of the Brazilian opossum, Monodelphis domestica. Monodelphis is a small pouchless marsupial whose young undergo a protracted period of postnatal development. Moreover, the Monodelphis retina represents a unique in vivo compartment for investigating cellular interactions that occur during early neural development and is an important system to study plasticity of neural stem cells following transplantation. Using bromodeoxyuridine (BrdU) labeling of newly generated cells, double-labeling immunohistochemistry and TUNEL labeling of apoptotic cells we have performed a detailed analysis of cell birth and death in the Monodelphis retina from fetal development through early postnatal life. Pregnant opossums or pups received a single injection of BrdU between gestational day 12 and postnatal day 35 (35PN), eyes were collected two hours after injection or on day 15, 30, or 60 of postnatal life. BrdU-labeled cells were visualized immunohistochemically. Cells were classified according to their morphology, location and immunoreactivity for cell-type specific antibodies. Cell genesis in the opossum retina begins at E13 and was near completion by 25PN. Apoptotic retinal cells were identified using the TUNEL technique for labeling of fragmented DNA. Apoptosis covered a relatively broad period of postnatal development, beginning around 10PN, peaking at 30PN, and concluding before 60PN. These results demonstrate that the retina of Monodelphis, a polyprotodont marsupial, is generated in a similar pattern to the wallaby, a diprotodont marsupial, and to eutherian species.

Oxygen consumption (VO 2 ), body temperature ( T b ) and wet thermal conductance ( C wet ), under resting conditions, exposure to low ambient temperature ( T a ) and during sustained exercise (treadmill running) were measured in three phylogenetic related (same family; Didelphidae) South American marsupials possessing similar body masses: Caluromys philander (arboreal/fruit and insect eating), Philander opossum (terrestrial and arboreal/omnivore), and Metachirus nudicaudatus (terrestrial/omnivore). Our measurements of VO 2 and C wet under resting conditions agree with those previously reported for other marsupials. We expected that C. philander would show a lower maximal sustained VO 2 , compared to the other two species, based on its more reduced skeletal muscle mass. However, the values obtained for C. philander were not statistically different (ANOVA) from those obtained for the other two species. When exposed to low ambient temperature (12 °C), differences among the three species were detected, i.e., M. nudicaudatus did not survive, while the other two species were able to reduce their T b under such conditions. C. philander gradually decreases its T b when cold exposed, and P. opossum shows a more pronounced T b drop only when exposure to low ambient temperatures occurs for a more prolonged period of time.

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.

Small animals must be capable of moving on a wide variety of surfaces; thus, examining the mechanics of locomotion on a wide variety of substrates is necessary to understand how the animal can utilize its habitat. Therefore, locomotor kinetics are examined on arboreal and terrestrial sloped substrates in the marsupial Monodelphis domestica (gray short-tailed opossum). Substrate reaction forces were measured as opossums moved across four trackways: 30 degrees upslope and 30 degrees downslope trackways, which were flat ("terrestrial") or cylindrical ("arboreal"). Regardless of substrate slope, medial limb forces were measured on arboreal trackways and usually lateral limb forces on terrestrial trackways. Otherwise the general patterns of vertical and craniocaudal forces and impulses were similar between same-sloped terrestrial and arboreal trackways. Some significant modifications to these gross patterns occurred: on the arboreal upslope trackway, hindlimbs supported more body weight than on the terrestrial uphill, possibly because hindlimbs were more stably positioned on the upslope arboreal trackway than forelimbs. Furthermore, the difference between fore- and hindlimbs with respect to craniocaudal impulses was less on the arboreal sloped trackways. In conclusion, kinetic patterns can usually be explained by body weight support roles and by the placement of the limbs on the arboreal trackway.

The biochemical composition of uterine and fetal fluids during pregnancy of the grey short-tailed opossum was compared with new and published data on the tammar wallaby. In the grey short-tailed opossum, there are three main phases of embryonic nourishment. During the first phase, the embryo obtains nutrients from uterine secretion transferred into the yolk sac. The amount of uterine secretion declines during the second phase up to the time of shell coat rupture. As a result, the protein concentration in yolk sac fluid also declines. During phase three, which begins with shell coat rupture, nutrients are predominantly available from the maternal blood. In the grey short-tailed opossum that lacks a vesicular, fluid-filled allantois, waste products such as urea are apparently stored in the yolk sac and from there pass into the maternal circulation across the invasive yolk sac placenta. In contrast, in the tammar wallaby, the main source of nutrients available to the late term fetus is glandular secretion that is complemented by substances from the maternal circulation via the chorio-vitelline placenta, and waste products are stored in the large, fluid-filled allantois.

The nucleotide sequence for pituitary prolactin cDNA from the marsupial bandicoot (Isoodon macrourus) was determined by reverse transcription-polymerase chain reaction and 5'/3' rapid amplification of cDNA ends. The deduced amino acid sequence showed high sequence identity with brushtail possum prolactin (95%) and all of the expected structural features of a quadruped prolactin. A prolactin gene tree was constructed and rates of evolution calculated for bandicoot, possum, opossum and several mammalian and non-mammalian prolactins. Bootstrap analysis provided strong support for marsupials as a sister group with eutherian mammals and weak support for opossum and bandicoot as an independent grouping from the brushtail possum. The rates of molecular evolution for marsupial prolactins were comparable to the slow rate seen in the majority of quadruped prolactins that have been sequenced.

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.

Opossums (Didelphis spp.) are the definitive host for the protozoan parasite Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). Opossums shed sporocysts in feces that can be ingested by true intermediate hosts (cats, raccoons, skunks, armadillos and sea otters). Horses acquire the parasite by ingestion of feed or water contaminated by opossum feces. However, horses have been classified as aberrant intermediate hosts because the terminal asexual sarcocyst stage that is required for transmission to the definitive host has not been found in their tissues despite extensive efforts to search for them [Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001b. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 95, 89-131]. In a 4-month-old filly with neurological disease consistent with EPM, we demonstrate schizonts in the brain and spinal cord and mature sarcocysts in the tongue and skeletal muscle, both with genetic and morphological characteristics of S. neurona. The histological and electron microscopic morphology of the schizonts and sarcocysts were identical to published features of S. neurona [Stanek, J.F., Dubey, J.P., Oglesbee, M.J., Reed, S.M., Lindsay, D.S., Capitini, L.A., Njoku, C.J., Vittitow, K.L., Saville, W.J., 2002. Life cycle of Sarcocystis neurona in its natural intermediate host, the raccoon, Procyon lotor. J. Parasitol. 88, 1151-1158]. DNA from schizonts and sarcocysts from this horse produced Sarcocystis specific 334bp PCR products [Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., MacKay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221-228]. Restriction fragment length polymorphism (RFLP) analysis of these PCR products showed banding patterns characteristic of S. neurona. Sequencing, alignment and comparison of both schizont and sarcocyst DNA amplicons showed 100% identity. Although Koch's postulates have not been demonstrated in this case study, the finding of mature, intact S. neurona schizonts and sarcocysts in the tissues of this single horse strongly suggests that horses have the potential to act as intermediate hosts. Further studies are needed to demonstrate Koch's postulates with repeated transfer of S. neurona between opossums and horses.

Chemical communication plays a major role in mammalian behaviour. One mammal with well-developed chemical senses is the grey short-tailed opossum, a small, South American marsupial that has become common in laboratory research. Male and female opossums scent-mark with different body parts, but apparently not with urine, and females enter oestrus following their exposure to male odours. To elucidate the effects of body odours on the behaviour of female opossums, we investigated their ability to discriminate between conspecific odours originating from different body parts. We simultaneously presented female opossums with two cotton balls smeared either with skin and glandular secretions or urine of conspecifics, or with distilled water or blank controls, and we analysed the time females spent investigating each stimulus. Odours of the male suprasternal gland elicited intensive investigation by the test females. Male flank and submandible odours, and female submandible odours, were also attractive to the females. Investigation of male and female urine was comparable to investigation of water and blank controls. We suggest that in this species, which inhabits semiarid areas, urine marking is avoided to reduce dehydration, and consequently urine has lost its biological significance as a chemosignal. On the other hand, glandular and skin secretions, which are more stable than aqueous deposits in dry conditions, allow the opossums to communicate with each other effectively and also to retain body fluids.

From April 1996 to December 2002 the prevalence of Sarcocystis neurona sporocysts in North American opossum (Didelphis virginiana) in Southern Michigan was estimated. Sporocysts of S. neurona were found in intestinal scrapings from 31 (15%) of 206 examined opossum. The frequency of infection was higher in adult animals (26/206; 12.6%) and females (19/206; 9.2%) than in juveniles (5/206; 2.4%) and males (12/206; 5.8%). Also, prevalence of S. neurona sporocysts in opossums in relation to factors such as age, sex, season, body condition, presence of concomitant infection, and presence of young in the pouch of females was studied in detail over the course of the year, 2002. Univariate analyses identified the following factors as being associated with the presence of S. neurona sporocysts in opossums: (i) for age, adult (odd ratio [OR] = 2.074, P = 0.0005); (ii) for sex, female (OR = 7.016, P = 0.0119); (iii) for season, summer (OR = 7.917, P = 0.0032) and spring (OR = 4.071, P = 0.1063); (iv) for body condition, poor (OR = 3.50, P = 0.1200) and good (OR = 1.167, P = 0.8637); (v) for the presence of concomitant infection (OR = 23.056, P = 0001), and (vi) for the presence of young in the pouch of females (OR = 40.083, P = 0.0001). Multivariate logistic-regression analyses selected the following factors as being significantly associated with presence of S. neurona sporocysts in opossums: (i) for the presence of concomitant infection (OR = 8.722, P = 0.0160) and (ii) for the presence of young in the pouch of females (OR = 31.915, P = 0.0065). The prevalence of S. neurona sporocysts in D. virginiana suggests that this opossum may constitute an ample reservoir of infection to other animals in the northern United States.

The Southern Pacific Rattlesnake (Crotalus helleri) is found in southwestern California (USA), southward through north Baja California (MX) into the northern part of southern Baja California (MX). In this study, the venoms from two Southern Pacific Rattlesnakes were characterized. The two venoms were different in color, concentration, and enzyme activities. Two commercial antivenoms neutralized both C. helleri venoms differently. Antivipmyn (Fab2H) and CroFab (FabO) neutralized both venoms but had different ED50. Four times more Fab2H antivenom was required to neutralize the C. helleri venom No. 011-084-009 than the venom from the snake No. 010-367-284. The hemorrhagic activity of two C. helleri venoms were neutralized differently by endothermic animal sera having a natural resistance to hemorrhagic activity of snake venoms. Opossums and Mexican ground squirrel sera did not neutralize the hemorrhagic activity of the venom No. 010-367-284. The sera of gray woodrats and hispid cotton rats neutralized all hemorrhagins in both C. helleri venoms. This is the first reported case in which opossum serum has not neutralized hemorrhagic activity of pit viper venom. Differences in the compositions of C. helleri venoms and their ability to be neutralized may help explain why snakebites are a difficult medical problem to treat and why effective polyvalent antivenoms are difficult to produce.

Our previous study morphologically revealed that the adult goat vomeronasal (VN) system was different from the rodent and opossum one, and at least two types of VN systems exist in mammals. However, it remains unknown whether the developments in both types of VN systems are ontogenetically distinct and when the goat VN system is established. In this study, we morphologically observed the fetal development of the goat accessory olfactory bulb (AOB) and VN neuron. In the fetus, Gi2-expressing VN terminals terminated at glomeruli throughout the AOB, and no immunoreactivities for Go were detected in the nerve terminals reaching into AOB. The layer structure of AOB rapidly developed in the latter half of gestation. In the VN organ (VNO), at the middle stage of gestation, the dendritic processes of VN neuron were exposed in the VN lumen, and scattered and thin microvilli existed on the protrusion of the VN neuron. In the apical part of dendritic processes, no clear vesicle existed. However, the immunohistochemistry of an olfactory marker protein (OMP) revealed that a few VN neurons with OMP exist in VN sensory epithelium (VSE) before birth, although marked immunoreactivities were detected in adult VSE. Fetal VN neurons appeared to be underdeveloped. These results suggest that the goat VN system is ontogenetically distinct from the rodent and opossum VN systems, and is underdeveloped before birth. The goat VN system will develop and mature during the early postnatal period similar to the rodent and opossum VN systems.

Gray short-tailed opossums (Monodelphis domestica) are small marsupials, which have recently become the subjects of numerous laboratory investigations. While these opossums have well-developed olfactory systems and complex scent-marking behaviors, the significance of their use of odors in conspecific communication is still poorly understood. Investigation of body odors by male and female opossums was examined in the present study. Males investigated flank and urine odors of nonestrous adult females significantly more than controls, but not urine from sexually inexperienced juvenile females or urine of females at cytological estrus. Since in this species females have an induced estrus, it would be advantageous for males to investigate and follow the odors of urine of diestrous females, which become receptive in proximity to males. Female opossums investigated odors of male mandibles and suprasternal glands significantly more than controls but not odors of male urine. We suggest that the use of glandular secretions is more common and more effective than urine for intraspecific communication between gray short-tailed opossums: In the semiarid conditions inhabited by the opossums, glandular secretions are less volatile and are effective for longer periods than urine and would be of greater value in intraspecific communication if, as suggested in the literature, these opossums are nomadic and meet one another infrequently.