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[Display omitted] • The basic neuroanatomy of the hypothalamus is highly conserved across all mammalian species. • Adult neurogenesis occurs in the hypothalamus of many mammalian species, including marsupials, but its rate is low. • Newly generated neurons are incorporated into the neural circuits of the hypothalamus, thereby inducing brain plasticity. In mammals, adult neurogenesis was first demonstrated in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampal formation. Further research showed that adult neurogenesis persists in other brain structures, such as the cerebral cortex, piriform cortex, striatum, amygdala, and hypothalamus. However, the origin of newly generated cells in these structures is not clear. Accumulating evidence indicates that newly generated neurons in the striatum or amygdala are derived from the SVZ, while in the adult hypothalamus, the proliferation of progenitor cells occurs in the ependymal cells lining the third ventricle, which give rise to new neurons. The heterogeneous cellular organization of the ependymal layer of the hypothalamus leads to different conclusions regarding the type of hypothalamic progenitor cells. In addition, adult hypothalamic neurogenesis occurs at low levels. Based on comparative and functional approaches, we synthesize the knowledge of newly generated cells in the adult hypothalamus. The aim of this review is to provide new insights on adult neurogenesis in the mammalian hypothalamus, with particular attention given to marsupial species. We highlight the number of adult-born neurons in various hypothalamic nuclei, debating whether their low number has an impact on hypothalamic function. [ABSTRACT FROM AUTHOR]

Oral rabies vaccination (ORV) is the primary tool for landscape level control and elimination of rabies virus in terrestrial wildlife species. However, there is currently a limited understanding of how different baiting strategies influence raccoon (Procyon lotor) uptake of ORV baits in non-agricultural habitats in the Southeastern United States, which may hinder the refinement of ORV management for raccoons in this region. Using a combination of new and existing data, we investigated the role of baited area (0.16 vs 3 km2), bait density (75 vs 150 baits/km2) and season (spring vs fall) on placebo ORV bait uptake by raccoons in four rural, non-agricultural habitats in the southeastern United States (bottomland hardwood forest, upland pine forest, riparian forest, and isolated wetlands). Increasing the baited area to 3 km2 increased the proportion of raccoons that consumed baits by 140 % in riparian forests and decreased the proportion consuming baits by 70 % in bottomland hardwood forests. A greater proportion of raccoons consumed baits in riparian habitat in spring compared to fall, but bait density did not influence uptake in either season in this habitat. Increasing the bait density during spring in bottomland hardwoods increased the proportion of raccoons that consumed baits by 270 %, but there was no effect of increasing bait density in bottomland hardwoods during fall. We suggest that variation in habitat contiguity and seasonal resource availability influence how raccoons utilize these habitats which in turn impacts habitat-specific ORV bait uptake. The estimated proportion of raccoons that consumed baits did not exceed 60 % for any treatment, and for most treatments was less than 40 %. These low uptake rates indicate a need for research into additional strategies to maximize raccoon uptake of ORV baits across rural, non-agricultural southeastern landscapes. • Role of oral rabies vaccine (ORV) bait density, area, and season on raccoon uptake varied by habitat. • Variation in bait uptake likely results from differences in how raccoons utilize these habitats. • Raccoons consumed more ORV baits than opossums, but uptake was low overall. [ABSTRACT FROM AUTHOR]

Summary The opossum (family Didelphidae) is a marsupial endemic to the Americas. Apart from the South American short-tailed opossum ( Monodelphis domestica ) and the Virginia opossum ( Didelphis virginiana ), there is considerable lack of knowledge about the health and diseases of most opossum species. Among these, the big-eared opossum ( Didelphis aurita ) is found in Argentina, Brazil and Paraguay. Natural and experimental studies have shown this species to be susceptible to infectious agents with zoonotic potential and the animals may play a role in transmission of such agents. However, neoplasia appears to be uncommon in this species. We describe the gross, microscopical and immunohistochemical features of a parotid salivary gland basal cell adenocarcinoma in a free-living big-eared opossum. This case represents the first report of salivary gland neoplasia in opossums. [ABSTRACT FROM AUTHOR]

• Detection of Babesia sp. belonging to "South American Marsupialia" piroplasmid clade;. • Detection of a putative novel Ehrlichia genotype in opossums;. • First molecular detection of hemoplasmas in D. marsupialis ;. • A putative new genotype closely related with Hepatozoon felis was detected in opossums;. • Copositivity between different agents was found in three opossums. Opossums are synanthropic marsupials able to interchange among wild, peri‑urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade. [ABSTRACT FROM AUTHOR]

Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons ( Procyon lotor ) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better studied and manipulated using in vitro and in vivo systems and compare the biology of two independent isolates. This study utilized culture-derived parasites from S. neurona strains derived from a raccoon or from a horse to initiate raccoon infections. Raccoon tissues, including fresh and cryopreserved tissues, were used to establish opossum ( Didelphis virginiana ) infections, which then shed sporocyts with retained biological activity to cause encephalitis in mice. These results demonstrate that sarcocysts can be generated using in vitro -derived S. neurona merozoites, including an isolate originally derived from a naturally infected horse with clinical EPM. This study indicates the life cycle can be significantly manipulated in the laboratory without affecting subsequent stage development, allowing further purification of strains and artificial maintenance of the life cycle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: This study was designed to investigate the morphology and distribution of argyrophil, argentaffins, and insulin-immunoreactive endocrine cells in the large intestine of the opossum Didelphis aurita. Fragments of the large intestine of 10 male specimens of the opossum D. aurita were collected, processed, and submitted for histochemistry, immunohistochemistry, and scanning electron microscopy. The tunics of the large intestine of D. aurita presented morphological characteristics that have already been described for eutherian mammals. The morphometric data showed that the inner circular layer of all portions and regions analyzed is thicker relative to the longitudinal layer, and these layers in the rectum are thicker compared to the cecum and ascending colon. The majority of mucus-secreting cells have acid and neutral mucins, suggesting that the production of mucus is mixed. The number of these cells increases in the region of the cecum toward the rectum. Important findings include the occurrence of argyrophil, argentaffins, and insulin-immunoreactive endocrine cells in all segments of the large intestine of the opossum (D. aurita). To the best of our knowledge, this is the first report about the presence of insulin-immunoreactive endocrine cells in the large intestine of the opossum (D. aurita). [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The aim of this study was to identify and quantify the argyrophil, argentaffin and insulin-immunoreactive cells (IIC) in the small intestine of the opossum Didelphis aurita. Seven adult male specimens of opossums were investigated. The animals were captured, and their blood insulin levels were determined. After euthanasia, fragments of the small intestine were processed for light microscopy and transmission electron microscopy, and submitted to histochemistry and immunohistochemistry for identification of argyrophil and argentaffin endocrine cells, and IIC. Argyrophil and argentaffin cells were identified in the intestinal villi and Liberkühn crypts, whereas IIC were present exclusively in the crypts. Ultrastructure of the IIC revealed cytoplasmic granules of different sizes and electron densities. The numbers of IIC per mm2 in the duodenum and jejunum were higher than in the ileum (p <0.05). The animals had low levels of blood insulin (2.8±0.78μIU/ml). There was no correlation between insulin levels and the number of IIC in the small intestine. The IIC presented secretory granules, elongated and variable morphology. It is believed that insulin secretion by the IIC may influence the proliferation of cells in the Liberkühn crypts, and local glucose homeostasis, primarily in animals with low serum insulin levels, such as the opossum. [Copyright &y& Elsevier]

Abstract: 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 9days postnatal cervical spinal cords regenerate, at 12days 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 8days and/or down-regulated by injury at 13days were genes known to promote growth, such as Mitogen-activated protein kinase kinase 1 or transcription factor TCF7L2. By contrast, at 13days, 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 12day 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. [Copyright &y& Elsevier]

Abstract: Gnathostoma turgidum is a gastric nematode parasite of opossums found in the Americas. We recently found that G. turgidum juveniles appear in the liver of the opossums where they become mature adults and almost synchronously move to the stomach during certain months of the year, suggesting the importance of the liver for the growth and maturation of this species in the final hosts. In this study we attempted to detect G. turgidum larvae in the liver of opossums, Didelphis virginiana that are the natural final hosts. The results show that tiny (<3mm in length) third stage larvae (L3) appeared in the liver of opossums around November and December. Also in the liver, we found large L3 of up to about 10mm in length together with juveniles and mature adults from February to March. In spite of their length, large L3 have 4 rows of hooklets, and their gonads remained undeveloped. Morphological features of the small and large L3 of G. turgidum are described including scanning electron microscope images. The seasonal switching of the several growth stages of G. turgidum from small L3 to adult worms in the liver and eventual migration to the stomach in opossums suggests the unique feature of G. turgidum utilizing the liver as the maturation site. [ABSTRACT FROM AUTHOR]

Abstract: The B cell receptor (BCR) is a multiprotein complex that is pivotal to antigen recognition and signal transduction in B cells. It consists of an antigen binding component, membrane Ig (mIg), non-covalently associated with the signaling component, a disulphide-linked heterodimer of CD79a and CD79b. In this study, the gene and corresponding cDNA for CD79a and CD79b in the gray short-tailed opossum, as well as the cDNA sequences for CD79a and CD79b in the tammar wallaby, are described. Many of the structural and functional features of CD79a and CD79b were conserved in both marsupials, including the ITAM regulatory motif in the cytoplasmic tails of both subunits. The marsupial CD79 sequences shared a high degree of amino acid identities of 76% (CD79a) and 72% (CD79b) to each other, as well as 60–61% (CD79a) and 58–59% (CD79b) with their eutherian counterparts. RT-PCR analysis of CD79a and CD79b transcripts in the immune tissues of tammar pouch young revealed CD79a transcripts in the bone marrow, cervical thymus and spleen at day 10 postpartum. CD79b transcripts were detected in the bone marrow and cervical thymus at day 10 but were not detected in the spleen until day 21 postpartum. These results suggest that a functional BCR may not be assembled until day 21 postpartum and the tammar neonate may not be capable of mounting an effective adaptive immune response until this time. The molecular information presented here will allow further investigation of the role of the CD79 subunits in marsupial B cell signaling, especially during ontogeny and disease. [Copyright &y& Elsevier]

BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron–exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals. [Copyright &y& Elsevier]

Abstract: 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). [Copyright &y& Elsevier]

Abstract: Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n =2 or 3) of each species were inoculated with 1×106 culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) – raccoon), TcI (NA – opossum), TcIIb (South American – human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7days post inoculation) than opossums (10days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts. [Copyright &y& Elsevier]

Abstract: Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals. [Copyright &y& Elsevier]

Abstract: CD5 has previously been identified in marsupial tissues using anti-human CD5. However, despite the known cross-reactivity of the antibody in marsupial tissues, the cDNA sequence has not previously been characterised in any marsupial. This study has identified the CD5 gene in the opossum genome database and has characterised the CD5 cDNA sequence from the tammar wallaby. Both marsupial CD5 sequences have a high level of sequence identity to known eutherian CD5 sequences, are cysteine-rich and have identical structural motifs to their eutherian homologs. CD5 transcripts were strongly expressed in adult tammar wallaby spleen, mammary node and blood, and expressed at a lower level in liver, kidney and heart tissues. Characterisation of CD5 in marsupials allowed a comparison to the epitope sequence of anti-human CD5 and showed a high level of sequence identity. [Copyright &y& Elsevier]

Abstract: 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 CD8α and CD8β 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% (CD8α) and 57% (CD8β) to each other as well as 36–45% (CD8α) and 38–41% (CD8β) with their eutherian counterparts. In addition, many of the signature features of eutherian CD8α and CD8β 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 CD8α subunit is also conserved. Interestingly, the opossum CD8α and the tammar CD8β sequences have a truncated cytoplasmic tail. RT-PCR analysis of CD8α and CD8β 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 CD8α and CD8β 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Quadrupedal animals moving on arboreal substrates face unique challenges to maintain stability. The torque generated by the limbs around the long axis of a branch during locomotion may clarify how the animals remain stable on arboreal supports. We sought to determine what strategy gray short-tailed opossums (Monodelphis domestica) use to exert torque and avoid toppling. The opossums moved across a branch trackway about half the diameter of their bodies. Part of the trackway was instrumented to measure substrate reaction forces and torque around the long axis of the branch. Kinematic analysis was used to estimate the center of pressure of the manus and pes; from center of pressure and vertical and mediolateral forces, the torque generated by substrate reaction forces versus muscular effort could be determined. Forelimbs generated significantly greater torque than hindlimbs, which is probably explained by the greater weight-bearing role of the forelimbs. Fore- and hindlimbs generated torque in opposite directions because contralateral fore- and hindlimbs typically contacted the branch. Torque generated by muscular effort, however, was often in the same direction in both fore- and hindlimbs. The muscle-generated torque is likely the result of mediolateral movement of the center of mass caused by mediolateral undulation of the torso. These results bear an important implication for the study of arboreal locomotion: center of mass dynamics are at least as important as static positions. M. domestica is a good representative for a primitive mammal, and comparisons with arboreal specialists will shed light on how proficient arboreal locomotion evolved. [Copyright &y& Elsevier]

Abstract: The architecture of the amygdaloid complex of a marsupial, the opossum Didelphis aurita, was analyzed using classical stains like Nissl staining and myelin (Gallyas) staining, and enzyme histochemistry for acetylcholinesterase and NADPH-diaphorase. Most of the subdivisions of the amygdaloid complex described in eutherian mammals were identified in the opossum brain. NADPH-diaphorase revealed reactivity in the neuropil of nearly all amygdaloid subdivisions with different intensities, allowing the identification of the medial and lateral subdivisions of the cortical posterior nucleus and the lateral subdivision of the lateral nucleus. The lateral, central, basolateral and basomedial nuclei exhibited acetylcholinesterase positivity, which provided a useful chemoarchitectural criterion for the identification of the anterior basolateral nucleus. Myelin stain allowed the identification of the medial subdivision of the lateral nucleus, and resulted in intense staining of the medial subdivisions of the central nucleus. The medial, posterior, and cortical nuclei, as well as the amygdalopiriform area did not exhibit positivity for myelin staining. On the basis of cyto- and chemoarchitectural criteria, the present study highlights that the opossum amygdaloid complex shares similarities with that of other species, thus supporting the idea that the organization of the amygdala is part of a basic plan conserved through mammalian evolution. [Copyright &y& Elsevier]

Abstract: Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function. [Copyright &y& Elsevier]

Abstract: α-Defensins are essential molecules of the innate immune system that have broad spectrum antimicrobial activity against a range of bacteria and viruses. To date, α-defensins have only been identified in the Euarchontoglires branch of the mammals. This has led to speculation that α-defensins may be specific to this group, a somewhat surprising finding, given their importance in the immune system. The mammalian genome project provided us with the opportunity to search for α-defensins in previously unexamined mammalian superorders. Using hidden Markov model (HMM) profile searching, we report the discovery of α-defensins in the African savanna elephant, the lesser hedgehog tenrec, and the nine-banded armadillo genomes representing two of the most basal mammalian superorders, Afrotheria and Xenarthra. Furthermore, we identify an α-defensin-like gene in the gray short-tailed opossum, suggesting that α-defensins may have evolved much earlier than previously thought, before the divergence of placental mammals and marsupials approximately 130mya. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Marsupials lack brown adipose tissue, and therefore rely exclusively on other tissues for thermogenesis. To determine the magnitude of phenotypic plasticity of the liver in response to changing metabolic demand, gray short-tailed opossums (M. domestica) were exposed to thermoneutral (28 °C) or cold (9–12 °C) conditions continuously for 6 weeks. Half of each group was also endurance trained with a treadmill program during their respective temperature exposure. Mass specific summit metabolism (V˙O2summit) increased 11% following cold acclimation, though there was no significant main effect by training on V˙O2summit. To estimate the contribution of the liver to whole animal oxidative activity, we determined liver mass, mitochondrial volume density, and total mitochondrial volume. Relative liver mass was 48% greater in cold-acclimated animals, whereas training had no effect on liver mass. The stereological analysis of hepatocyte ultrastructure suggests the percentage of intracellular volumes remained unchanged in response to either aerobic challenge. Thus, following cold-acclimation, there is a 20% increase in the total mitochondrial volume of the liver. This increase could account for nearly half (44%) of the observed increase in whole animal V˙O2summit following cold exposure. [Copyright &y& Elsevier]

Esophageal peristalsis generally does not return to normal after surgical treatment of achalasia. Direct electrical stimulation of the vagus nerve is known to stimulate antegrade peristalsis in the normal esophagus; however, it is not known whether electrical stimulation will induce return of peristalsis once an achalasia-like disorder has been established. The objective of this study was to perform quantitive and qualitative measurements of motility during electrical stimulation of the vagal nerve in an animal model of achalasia. An already established and verified animal achalasia model using adult North American opossums (Didelphis virginiana) was used. Fifteen opossums were divided into three groups. Sham surgery was performed on three animals (group 1). In group 2 (n = 6) a loose Gore-Tex band (110% of the esophageal circumference) was placed around the gastroesophageal junction to prevent relaxation of the lower esophageal sphincter during swallowing. In group 3 (n = 6) a relatively tighter band (90% of the esophageal circumference) was used to further elevate the lower esophageal sphincter pressure. At 6 weeks, after manometric and radiolologic confirmation of achalasia, electrical stimulation of the esophagus was performed before and after removal of the band using a graduated square-wave electrical stimulus. Changes in esophageal neural plexi were assessed histologically. Pre- and postoperative manometric data were compared using standard statistical techniques. No difference was observed in esophageal characteristics and motility after sham surgery in group 1. Animals in group 2 demonstrated a vigorous variety of achalasia (high-amplitude, simultaneous, repetitive contractions), moderate esophageal dilatation, and degeneration of 40% to 60% of intramuscular nerve plexi. Animals in group 3 developed amotile achalasia with typical low-amplitude simultaneous (mirror image) contractions, severely dilated (“bird beak”) esophagus, and degeneration of 50% to 65% of nerve plexi. Vagal stimulation in group 2 demonstrated a significant increase in the amplitude of contractions (P<0.001) and return of peristaltic activity in 49% of swallows before band removal. After band removal, all of the contractions were peristaltic. In group 3 vagal stimulation before and after removal of the band demonstrated a significant increase in amplitude of contractions (P<0.0001) but no return of propagative peristalsis before band removal, however, 44% of contractions were progressive in the smooth portion of the esophagus after removal of the band. Electrical stimulation of the vagus nerve improved the force of esophageal contractions irrespective of the severity of the disease; however, peristaltic activity completely returned to normal only in the vigorous (early) variety of achalasia. Removal of the functional esophageal outlet obstruction, as with a surgical myotomy, may be necessary to obtain significant peristalsis with vagal pacing in severe achalasia. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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 μm and were separated from the host tissue by a thick (5–20 μm) 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). [Copyright &y& Elsevier]

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by infection of the central nervous system with the protozoan parasite Sarcocystis neurona. A herd-level analysis of a cross-sectional study of serum antibodies to S. neurona in Michigan equids was conducted, using data collected in 1997 for study that included 1121 equids from 98 Michigan horse farms. Our objective was to identify specific herd-level risk factors associated with seropositivity. We tested associations between herd seroprevalence and various farm-management practices (including feed-storage methods and wildlife control). Multivariable models were developed for three strata based on relative opossum abundance (opossum districts).Herd seroprevalence ranged from 0 to 100% (median=57%). No risk factor was significantly associated with herd seroprevalence at P≤0.05 in all opossum districts. Our results suggest that equids living in areas with large opossum populations might be infected with S. neurona from multiple sources. [Copyright &y& Elsevier]

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 45×25 μm 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. [Copyright &y& Elsevier]

A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system. [Copyright &y& Elsevier]

• Strain background contributes to disease susceptibility of Scid knockout mice to Sarcocytsis neurona encephalitis. • Interferon gamma derived from T cells is sufficient to protect mice from Sarcocytsis neurona encephalitis. • Interferon gamma plays an important role in Sarcocytsis neurona resistance. Sarcocystis neurona is the predominant etiological agent of the infectious equine neurologic disease, equine protozoal myeloencephalitis (EPM), which is prevalent in the United States. A wealth of knowledge about S. neurona biology and its life cycle has accumulated over the last several decades. However, much remains unknown about the aberrant equine host's immune response to S. neurona and the relatively high prevalence of exposure to the protozoa but relatively infrequent occurrence of clinical neurologic disease. Mouse models simulating EPM are commonly used to study the disease due to numerous challenges associated with studying the disease in horses. The critical role of the cytokine, interferon gamma (IFNγ), in protection against S. neurona encephalitis has been well established as Ifnγ−/− mice are highly susceptible to S. neurona encephalitis. However, there are discrepancies in the literature regarding S. neurona disease susceptibility in lymphocyte deficient mice, lacking T-lymphocytes and their associated Ifnγ production. In the current study, we investigated S. neurona encephalitis susceptibility in 2 genetically different strains of lymphocyte null mice, C57Bl/6 (B6). scid and Balb/c. scid. The B6. scid mouse was determined to be susceptible to S. neurona encephalitis as 100 % of infected mice developed neurologic disease within 60 days post infection (DPI). The Balb/c. scid mouse was nearly disease resistant as only 10 % of mice developed neurologic disease 60 DPI. Encephalitis was histologically demonstrable and S. neurona was identified in cerebellar samples collected from B6. scid but absent in Balb/c. scid mice. To further investigate the importance of T-lymphocyte derived Ifnγ, T- lymphocytes were adoptively transferred into B6. scid mice. The adoptive transfer of Ifnγ competent T- lymphocytes offered complete protection against S. neurona encephalitis but transfer of Ifnγ deficient T- lymphocytes did not with 100 % of these recipient mice succumbing to S. neruona encephalitis. Histological analysis of collected cerebellar samples confirmed the presences of S. neurona and encephalitis in recipient mice that developed neurologic disease. These studies show that the background strain is critical in studying SCID susceptibility to S. neurona disease and suggest a protective role of Ifnγ producing T- lymphocytes in S. neurona encephalitis susceptible mice. [ABSTRACT FROM AUTHOR]

• First report of Leptospira sp. in Central Nervous System of D. albiventris in the Brazil. • Phylogenetic analysis showed similarity with Leptospira interrogans sequences. • Leptospira interrogans DNA was detected only in CNS. 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. [ABSTRACT FROM AUTHOR]

Globally, hemotropic mycoplasmas (hemoplasmas) comprise an emerging or remerging bacteria group that attaches to red blood cells of several mammal's species and in some cases, causing hemolytic anemia. Herein, we assessed the occurrence, genetic diversity, the factors coupled to mammals infection, and the phylogeographic distribution of hemoplasmas in sylvatic and synanthropic mammals and their associated ectoparasites from Brazil. We collected spleen and/or blood samples from synanthropic rodents (Rattus rattus [ N = 39] and Mus musculus [ N = 9]), sylvatic rodents (Hydrochoerus hydrochaeris [ N = 14]) and opossums (Didelphis albiventris [ N = 43]). In addition, ticks (Amblyomma spp. [ N = 270] and lice (Polyplax spinulosa [ N = 6]) specimens were also sampled. Using a PCR targeting the 16S rRNA region, out of 48 small rodents, 14 capybaras and 43 opossums DNA samples, hemoplasma DNA was found in 25%, 50%, and 32.5% animals, respectively. Besides, we reported hemoplasma DNA in Amblyomma sp. (22.2% [2/9]) and lice (100% [2/2]) pools samples from rats, and one female A. sculptum DNA sample (3% [1/33]) obtained from a capybara. Additionally, and in agreement with ML analysis, the network analyses showed a clear phylogenetic separation among the hemoplasmas genotypes found in the different host species sampled, thus, suggesting the absence of cross-species hemoplasmas transmission between the mammals trapped. Finally, using the NTC network analysis, we reported the same 16S rRNA Mycoplasma genotype circulating in Rattus sampled in Brazil, Hungary, and Japan. [ABSTRACT FROM AUTHOR]