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Preliminary observations on experimental leprosy in tupaias (Tupaia belangeri yunalis).

The Tupaia belangeri yunalis (tree shrew) is one of the primitive primates. They were inoculated subcutaneously in the footpad or intravenously with Mycobacterium leprae from a patient with multibacillary leprosy. As controls, the footpads of CFW mice were inoculated with the same suspension of M. leprae. The results showed growth of acid-fast bacilli (AFB) in the footpads of locally inoculated CFW mice and in the footpads of both locally and intravenously inoculated tupaias. Whereas the numbers of AFB declined in the footpads of CFW mice after 12 months, they increased in the tupaia footpads, up to 2.44 x 19(9) AFB/g of tissue. The footpads of one tupaia were swollen, which on section revealed a granulomatous infiltration, including foamy and heavily infected macrophages. M. leprae were also seen in the branches of cutaneous nerves. Also AFB occurred in some viscera. Preliminary studies indicate that the AFB multiplying in tupaias are M. leprae.

Animals

Experimental infection and the state of viral latency of adult tupaia with herpes simplex virus type 1 and 2 and infection of juvenile Tupaia with temperature-sensitive mutants of HSV Type 2.

The susceptibility of adult Tupaia Belangeri to infection with herpes simplex virus (HVS) was investigated. Adult animals were inoculated intraperitoneally with HSV type 1 or 2. With the exception of HSV-2, strain HG-52, 10(5)--10(6) PFU of all HSV strains caused lethal infection irrespective of the age of the animals. Infections HSV was recovered from the spinal cord of those animals which had survived infection with a low dose of virus. The DNA of the recovered viruses was compared to the DNA of the inoculated HSV. The viral genome of the recovered HSV was unchanged as judged by analysis of the fragment pattern of the viral DNA's using restriction endonucleases. Animals which had survived the first HSV infection were protected against a second infection even at highly lethal doses of HSV-1 or 2. Juvenile Tupaia survived infection with temperature-sensitive mutants of HSV-2, strain HG-52, which induced protection against a second infection with lethal doses of HSV-1 or 2.

Aging

Cryptococcosis in tree shrews (Tupaia tana and Tupaia minor) and elephant shrews (Macroscelides proboscides).

Fungal infections due to Cryptococcus neoformans occurred in seven short-eared elephant shrews (Macroscelides proboscides), six large tree shrews (Tupaia tana), and five lesser tree shrews (Tupaia minor) at the National Zoological Park during a 30-mo period in 1991-1993. Clinical signs were absent or included weight loss, shivering, dyspnea, and/or neurologic disease. Definitive antemortem diagnostic techniques included tracheal lavage and serum cryptococcal antigen latex agglutination titers. Thirteen cases were diagnosed solely by postmortem examination. The source of infection for these animals was uncertain, but C. neoformans is commonly found in soil and other organic material. Two lesser tree shrews and one large tree shrew received antifungal therapy and converted to a negative serum cryptococcal antigen titer.

Animals

[The infection of hepatitis D virus in adult tupaia].

A new animal model of HDV/HBV infection in adult tupaia (tree shrews) was established to study its pathogenesis and treatment of hepatitis D. In 20 artificially fed adult tupaiae, the positive rate of serum HBsAg was 75% (15/20) after inoculation of human HBV DNA positive serum. After inoculation with positive human serum HBV DNA and positive HDV RNA by way of coinfection and superinfection, serum HBsAg, HDAg and anti-HD were positive one after another in 8 out of 13 tupaiae of the coinfection group and in 6 out of 9 tupaiae of the superinfection group. In some of them HDV RNA was positive in serum or in liver tissue. Transmission of HDV/HBV infection among tupaiae was successful. In group I, animals were inoculated with human HDV/HBV positive serum. In Group II and III, animals were inoculated with tupaia HDV/HBV positive serum of the Group I and II respectively. Serum HBsAg, HDAg and anti-HD became positive one after another in 3 out of 4 tupaiae of the Group II and 4 out 5 tupaiae of the group III. In 2 animals of the Group III, serum HDV RNA was positive and HBsAg and HDAg were detected in the liver tissue. Serum HBsAg was persistantly positive for 16-19 months in the superinfection group. In 2 of them, serum HDAg and anti-HD were alternatively positive and in one of them serum anti-HD was positive persistantly indicating that HDV infection may persist and transform into chronic HDV infection. In the HDV infected tupaiae, there were elevated serum ALT and hepatitis-like pathological changes in the liver tissue which were similar to those in the chimpanzee. We conclude that tupaia may be used as an experimental model of hepatitis D.

Animals

Studies on the juxtaglomerular apparatus. VI. Sympathetic innervation, catecholamines and the renin-angiotensin-system in rats and tree-shrews (Tupaia belangeri).

It has previously been reported that the primitive primate Tupaia belangeri develops a renal failure when exposed to psychosocial stress. In order to learn if this high susceptibility to stress of the Tupaia kidney can be correlated with morphological and functional parameters of the juxtaglomerular apparatus (JGA) and the renin-angiotension system, comparative experiments were performed on Tupaia and rat. Our results reveal an outstandingly high potency of the JGA and the renin-angiotensin system in Tupaia as evident from the following findings: The Tupaia JGA contains a great number of epithelioid cells abounding in renin granules (electron microscopy). The renin content of the Tupaia kidney is considerably higher than in the rat (radio-immunoassay). The sympathetic innervation of the kidney and especially of the JGA is abundant in Tupaia (fluorescence and electron microscopy). Catecholamine contents of the kidney and other organs are significantly higher in Tupaia than in rats (spectrophotofluorometry). Our results support the previously developed concept of a potent intrarenal neuroendocrine interaction at the JGA level favouring, under certain conditions of social stress, the development of acute renal failure in Tupaia belangeri.

Angiotensins

Tree shrew (Tupaia) herpesviruses.

Five Tupaia herpesviruses have been isolated until now: four in our laboratory which were termed THV-2, 3, 4, and 5, whereas THV-1 has been isolated by Melnick and his colleagues. THV-2 was isolated from tumour cell culture of a high-grade malignant lymphoma of a Tupaia, THV-3 was released from a cell culture of another Tupaia lymphoma, THV-4 from a spleen tissue culture of a moribund animal with finely granulated liver cirrhosis, and THV-5 from cultured spleen cells of an apparently healthy tree shrew. THV-1 to 5 were efficiently propagated, plaque-purified and cloned on Tupaia embryonic fibroblasts. The five isolates of Tupaia herpesviruses are easily distinguished from each other by restriction enzyme analysis of their genomes. THV-1 to 4 are highly pathogenic (lethality 100%) for juvenile Tupaias by intravenous inoculation. In contrast, only 25% lethality was found by intraperitoneal administration. THV-1 to 4 can persist as a latent infection in spleens of Tupaias and rabbits, which allows the recovery of infectious virus from cultured spleens of both animals. THV-2 and 3 induced hyperplasia of the thymus of rabbits which developed malignant thymoma in a few cases. The biological properties and genomic size and structure indicate that THV cannot be considered to belong to one of the three existing subfamilies of herpesviruses.

Animals

Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo.

For the systematic analysis of various clinical and molecular aspects of hepatitis B virus (HBV) infection, an experimental small animal system of HBV infection would be a great advance. The susceptibility to HBV infection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infection results in viral DNA and RNA synthesis in hepatocytes and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute, self-limited hepatitis B in humans HBsAg is rapidly cleared from serum, followed by seroconversion to anti-HBe and anti-HBs. These data clearly tht HBV is infectious to tupaia hepatocytes in vitro and transiently in vivo. Tupaias, therefore, may become a useful model for the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, the steps involved in hepatocarcinogenesis as well as the evaluation of various antiviral strategies.

Animals

The two-oscillator circadian system of tree shrews (Tupaia belangeri) and its response to light and dark pulses.

The wheel-running activity rhythm of tree shrews (tupaias; Tupaia belangeri) housed in constant darkness (DD) phase-advanced following a 3-hr light pulse at circadian time (CT) 21. Dark pulses of 3 hr presented to tupaias in bright constant light (LL) did not induce significant phase shifts of the free-running activity rhythm, irrespective of the CT. In dim LL, tupaias showed simultaneous splitting of their circadian rhythm of wheel-running activity, nest-box activity, and feeding behavior. Light pulses of 6 hr and 2300 lux were presented to 13 tupaias with split wheel-running activity rhythms. These light pulses induced immediate phase shifts in the two components of the split rhythm in opposite directions. No differences were observed between the light-pulse phase response curves of the two components. Equally large immediate phase advances were induced in both components by light pulses of 230 lux, but not by 23 lux. The final phase shifts were small at all CTs. In two tupaias, activity rhythms transiently split and re-fused. Analysis of the relative position of the components in one of these indicates asymmetry in the coupling between the components.

Animals

Morphogenesis of megamitochondria in the retinal cone inner segments of Tupaia belangeri (Scandentia).

The morphogenesis of the megamitochondria in the retinal cones of prenatal, young postnatal and adult tree shrews (Tupaia belangeri) was studied by transmission electron microscopy and three-dimensional reconstruction techniques. The initial assembly of the supranuclear cone mitochondria and their subsequent migration towards the developing inner segment conform to the morphogenetic pattern known from other mammals. Within the first postnatal week, however, a marked increase in both the number of the cristae and the matrix density occurs in the inner segment mitochondria of Tupaia. These mitochondria then grow, initially exhibiting a basal-to-apical size-gradient. In the 17-day-old Tupaia, this gradient is superseded by a radial size-gradient that, in addition to the single apical megamitochondrion, is characteristically found in the adult Tupaia. The number of megamitochondria remains almost constant from day 12 of postnatal ontogenesis to the adult stage. Each megamitochondrion consists of an apically located body from which several long processes project towards the base of the inner segment. In the older stages, the number of small mitochondria that most probably have budded off from the megamitochondrial processes clearly increases. We consider that megamitochondria in the cone inner segments of Tupaia arise by the growth of a single mitochondrion and not by the fusion of smaller mitochondria.

Animals

Demonstration in Tupaia papillary muscle preparations of alpha-adrenoceptors mediating positive inotropic effects: comparison with guinea-pigs.

1. Positive inotropic responses to alpha- and beta-adrenoceptor agonists of isolated papillary muscles from the tree shrew (Tupaia), were compared with those from guinea-pigs. 2. In Tupaia, the concentration-response curve for phenylephrine, unlike that for isoprenaline, was not affected by pindolol in a concentration (10(-8) M) sufficient to block beta-adrenoceptor-mediated responses, but it was significantly shifted to the right by phentolamine (10(-6) M). In guinea-pig papillary muscles, however, the concentration-response curve for phenylephrine, like that for isoprenaline, was shifted to the right by pindolol (10(-8) M) but was unaltered by phentolamine (10(-6) M). Furthermore, when the mean concentrations of agonists inducing maximal positive inotropic responses were compared (relative to that of isoprenaline = 1.0), phenylephrine was found to be only slightly less potent (0.84 +/- 0.04; n = 5) in Tupaia and much less potent (0.33 +/- 0.06; n = 5) in the guinea-pig. 3. Although in Tupaia papillary muscles the increase in developed tension induced by a combination of phenylephrine and isoprenaline did not significantly differ from that by phenylephrine alone, it was approximately 3 times larger than that produced by phenylephrine alone in guinea-pigs. 4. These results indicate that in papillary muscles from Tupaia, unlike the guinea-pig, the positive inotropic effects of phenylephrine can be mediated by alpha-adrenoceptors.

Adrenergic alpha-Agonists

Unusual nature and possible evolutionary implications of the male vesicular gland secretion in the tree shrew, Tupaia glis.

BACKGROUND: Whereas the secretion of the male vesicular gland in most mammals is amorphous, that of the tree shrew, Tupaia glis, was observed to be stored as globules. METHODS: Vesicular and prostate glands from Tupaia, fixed in glutaraldehyde and osmium, were studied in the light and electron microscopes. Other materials considered included the Tupaia ejaculate produced by electroejaculation and, for comparative purposes, sections of the vesicular gland from a dermopteran, the flying lemur. RESULTS: The vesicular gland epithelium in Tupaia secretes small granular aggregates and occasionally a denser aggregate that is associated with cells having obvious apical Golgi lamellae. In the alveolar lumen, these aggregates unite with others to form, respectively, granular and some dense globules of up to approximately 15 mu in diameter, which appear as such in semen produced by electroejaculation. In contrast to the prostate, however, precursor secretion vesicles were rare in the vesicular epithelium. Although poorly fixed, the vesicular gland secretion from a flying lemur also appeared to form globules. CONCLUSIONS: Although it is unlike the homogeneous secretion elaborated in most mammals, including primates and insectivores, the globular product of the Tupaia vesicular gland seems comparable to that in a variety of mega- and microbats, among representative species of which it appears to provide the bulk material for the vaginal copulation plug. Because a museum specimen examined here also indicates its occurrence in a flying lemur, the globular vesicular gland secretion common to Tupaiidae, to at least some Mega- and Microchiroptera, and apparently to Dermoptera may provide a soft tissue feature of some value in the cladistic approach to phylogenetic reconstruction within the Archonta. Anat.

Animals

Terminations of individual optic tract fibers in the lateral geniculate nuclei of Galago crassicaudatus and Tupaia belangeri.

The morphology and laminar distribution of individual optic fibers projecting to the lateral geniculate nucleus (GL) of Galago and Tupaia were studied following iontophoretic injections of horseradish peroxidase (HRP) into the optic tract. In Galago the GL is composed of three functionally matched pairs of layers, each characterized by cells of a given size, one large, one medium-sized, and one small. The results show that there is a close correspondence between the size of the afferent fibers and the size of the neurons in the target layer: large axons project to the magnocellular layers, medium-sized axons project to the parvicellular layers, and small fibers project to the intercalated layers. In Tupaia the GL is composed of two functionally matched pairs and two unmatched layers. Optic fibers that project to the medial matched pair (1 and 2) are only slightly larger than those that project to the lateral matched pair (4 and 5), but both are larger than those that project to the unmatched layers (3 and 6). In both species terminal arbors and the distribution of terminal boutons within layers corresponded closely with the organization of dendritic processes of cells in the target layer. This correspondence was particularly evident in the parvicellular layers in Galago and in layer 6 in Tupaia: parvicellular terminal arbors, like the dendrites of parvicellular cells, are organized in narrow columns oriented along lines of projection, whereas layer 6 terminal arbors, like the dendrites of layer 6 cells, are oriented in elongated strips perpendicular to lines of projection. In both species there was evidence for sublaminar terminations in some layers. These were restricted to the parvicellular layers in Galago and layers 4 and 5 in Tupaia. With the exception of a small number of fine fibers in the intercalated layers in Galago, optic fibers in both species terminated in one and only one layer in a set. The significance of this result depends on the relation between ganglion cell classes and what is being segregated in different GL layers. Lateral geniculate lamination varies even in closely related species and has evolved independently in such distantly related lines as carnivores and primates. It is not surprising, therefore, that what is being segregated varies from species to species.

Animals

Rod receptors in the retina of Tupaia belangeri.

The retina of Tupaia belangeri was studied by transmission electron microscopy. - Among a vast majority of cones a few typical rods have been found as a distinctly different class of photoreceptors. The outer segments of the rods measure 10 microns X 1.5 microns versus 6 microns X 2 microns in the cones. The repeating unit of the rod discs is 18 nm versus 22 nm in the cone discs. Rod discs tend to lose their continuity with the ciliary membrane, while cone discs do not. Rods lack the highly specialized giant mitochondria of the cone inner segment of Tupaia. The rods of Tupaia show other distinctive characters of mammalian rods as well. However, the rods of Tupaia are relatively short and do not develop a rod fiber.

Animals

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.

Animals

An X-Y homologous pairing segment in tree shrews (Tupaia).

High-resolution early replication banding of tupaia metaphase chromosomes revealed a synchronous early replicating segment in the short-arm telomeric regions of the active and inactive X chromosomes and in the long-arm telomeric region of the Y chromosome. Hybridization with the human-derived pseudoautosomal probe 113F (STIR) showed that this repeat is conserved and specifically localized within these synchronously early replicating segments of the X short arm and the Y long arm of all three tupaia species (Tupaia belangeri, T. chinensis, and T. glis) investigated. Moreover, meiotic studies demonstrated that a synaptonemal complex is formed at one telomeric end of the XY bivalent during the pachytene stage of meiosis in a male T. glis specimen. Thus, apart from the mouse, the tupaias are the first nonprimate mammals for which cytogenetic and molecular evidence is provided that their highly heteromorphic X and Y chromosomes share a conserved homologous segment in the telomeric position, a location that is compatible with pairing and crossing-over in male meiosis. Taken together, these observations strongly, albeit indirectly, suggest that this chromosome segment at the tip of a sex-chromosome arm might behave pseudoautosomally.

Animals

Dorsal root afferents to clarke's column from hindlimb cord levels in Tupaia and Galago.

The projection of hindlimb dorsal root afferents to Clarke's column has been studied in the tree shrew (Tupaia glis) and lesser bushbaby (Galago senegalensis). In the bushbaby, fibers from K12, L1 and L3 projected ipsilaterally to Clark's nucleus from levels L3 to T3, L2 to T4 and L3 to T5 respectively. In Tupaia, afferents from T11, L1, and L3 terminated ipsilaterally within the nucleus from segments L2 to T4, L3 to T6, and L3 to T8. Fibers from T12, L1, and L3 in Galago and T11 in Tupaia terminated within the even to lateral aspect of the nucleus at lower levels, throughout the nucleus at middle levels and in the dorsal aspect of the column at rostral levels. Fibers from T11 and L1 have a similar ventrolateral pattern of termination in lower levels, however, at rostral levels preterminal debris was present throughout the width of the nucleus. Fibers from L1 and L3 in the bushbaby form a complex longitudinal network with the medullary region of the nucleus in segments T11 to T8. Afferents from levels T5 in Tupaia and L6 in Galago projected ipsilaterally to the nucleus from level L3 to T10, and T8, respectively. Fibers from these segments terminated throughout the extent of the column at L3 and, rostrally projected to more dorsal regions of the nucleus. In this study, fibers from S2, and S3 and CCO2 did not terminate in Clark's column. Both the segmental distribution of hindlimb dorsal root fibers and their pattern of termination in Clark's nucleus in the tree shrew were similar to that reported in quadrupedal primates and other quadrupedal mammalian forms. The results of this study were interpreted as evidence which relates the complex organization of the Clark column system in the lesser bushbaby to its vertical clinging and leaping style of locomotion.

Animals