Expanding the Arterivirus Host Spectrum: Olivier's Shrew Virus 1, A Novel Arterivirus Discovered in African Giant Shrews.

The family Arteriviridae harbors a rapidly expanding group of viruses known to infect a divergent group of mammals, including horses, pigs, possums, primates, and rodents. Hosts infected with arteriviruses present with a wide variety of (sub) clinical symptoms, depending on the virus causing the infection and the host being infected. In this study, we determined the complete genome sequences of three variants of a previously unknown virus found in Olivier's shrews (Crocidura olivieri guineensis) sampled in Guinea. On the nucleotide level, the three genomes of this new virus, named Olivier's shrew virus 1 (OSV-1), are 88-89% similar. The genome organization of OSV-1 is characteristic of all known arteriviruses, yet phylogenetic analysis groups OSV-1 separately from all currently established arterivirus lineages. Therefore, we postulate that OSV-1 represents a member of a novel arterivirus genus. The virus described here represents the first discovery of an arterivirus in members of the order Eulipotyphla, thereby greatly expanding the known host spectrum of arteriviruses.

Serological and PCR investigation of Yersinia pestis in potential reservoir hosts from a plague outbreak focus in Zambia.

BACKGROUND: Plague is a bacterial zoonotic disease, caused by Yersinia pestis. Rodents are the natural hosts with fleas as the vehicle of disease transmission. Domestic and wild dogs and cats have also been identified as possible disease hosts. In Zambia, plague outbreaks have been reported in the Southern and Eastern regions in the last 20 years. Based on these observations, Y. pestis could possibly be endemically present in the area. METHODS: To substantiate such possibility, sera samples were collected from rodents, shrews, dogs and cats for detection of antibodies against Fraction 1 gene (Fra1) of Y. pestis while organs from rodents and shrews, and fleas from both dogs and rodents were collected to investigate plasminogen activator gene (pla gene) of Y. pestis using ELISA and PCR respectively. RESULTS: A total of 369 blood samples were collected from domestic carnivores, shrews and domestic and peri-domestic rodents while 199 organs were collected from the rodents and shrews. Blood samples were tested for antibodies against Fra1 antigen using ELISA and 3% (5/165) (95% CI 0.99-6.93%) dogs were positive while all cats were negative. Of 199 sera from rodents and shrews, 12.6% (95% CI 8.30-17.98%) were positive for antibodies against Fra1 using anti-rat IgG secondary antibody while using anti-mouse IgG secondary antibody, 17.6% (95% CI 12.57-23.60%) were positive. PCR was run on the organs and 2.5% (95% CI 0.82-5.77%) were positive for plasminogen activator gene of Y. pestis and the amplicons were sequenced and showed 99% identity with Y. pestis reference sequences. All 82 fleas collected from animals subjected to PCR, were negative for pla gene. The specific rat-flea and dog-flea indices were 0.19 and 0.27 respectively, which were lower than the level required to enhance chances of the disease outbreak. CONCLUSIONS: We concluded that plague was still endemic in the area and the disease may infect human beings if contact is enhanced between reservoir hosts and flea vectors. The lower specific rodent-flea Indices and absence of Y. pestis in the potential vectors were considered to be partly responsible for the current absence of plague outbreaks despite its presence in the sylvatic cycle.

The common shrew (Sorex araneus): a neglected host of tick-borne infections?

Although the importance of rodents as reservoirs for a number of tick-borne infections is well established, comparatively little is known about the potential role of shrews, despite them occupying similar habitats. To address this, blood and tick samples were collected from common shrews (Sorex araneus) and field voles (Microtus agrestis), a known reservoir of various tick-borne infections, from sites located within a plantation forest in northern England over a 2-year period. Of 647 blood samples collected from shrews, 121 (18.7%) showed evidence of infection with Anaplasma phagocytophilum and 196 (30.3%) with Babesia microti. By comparison, of 1505 blood samples from field voles, 96 (6.4%) were positive for A. phagocytophilum and 458 (30.4%) for Ba. microti. Both species were infested with the ticks Ixodes ricinus and Ixodes trianguliceps, although they had different burdens: on average, shrews carried almost six times as many I. trianguliceps larvae, more than twice as many I. ricinus larvae, and over twice as many nymphs (both tick species combined). The finding that the nymphs collected from shrews were almost exclusively I. trianguliceps highlights that this species is the key vector of these infections in this small mammal community. These findings suggest that common shrews are a reservoir of tick-borne infections and that the role of shrews in the ecology and epidemiology of tick-borne infections elsewhere needs to be comprehensively investigated.

Testis size, sperm characteristics and testosterone concentrations in four species of shrews (Mammalia, Soricidae).

The aim of this study was to establish and compare the sperm characteristics in four shrew species in the context of the sperm competition hypothesis. As expected, the large relative testis size in promiscuous species was associated with a high number of cauda epididymal spermatozoa and a high concentration of circulating testosterone. In addition, in Sorex and Neomys, species with high intensity of sperm competition, the spermatozoa stored in cauda epididymis were characterized by high percentage of progressive motility whereas in Crocidura and Suncus, the cauda epididymal spermatozoa were motile but with very low percentage of progressive motility. This capability is achieved only following the passage through the vas gland, a specialized region for sperm storage located along the vas deferens in these shrew species. The hypothesis that sperm competition is positively correlated with spermatozoa length could not be confirmed. In Crocidura and Suncus, the total sperm length is increased by the large sperm head due to a big acrosome. This trait, specific to the subfamily Crocidurinae, may results from a selective pressure independent of the context of sperm competition, related to a specific, but as yet unclear role, for the acrosome during the fertilization.

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula.

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.

Phylogeography of wild musk shrew (Suncus murinus) populations in Asia based on blood protein/enzyme variation.

The musk shrew (Suncus murinus) is an insectivore species that inhabits tropical and subtropical Asia widely. To clarify the genetic relationship among wild musk shrew populations, we examined the electrophoretic variants of biparentally inherited genetic markers at 10 loci coding for eight blood proteins/enzymes in a total of 639 animals and compared the results obtained from the mitochondrial DNA data. The principal-component analysis performed using the allele frequency data revealed that the 17 populations could be divided into two major groups, a South Asian group and a Southeast Asian group that includes several island populations bound by Myanmar. The degrees of genetic divergence among populations were higher within the Southeast Asian group than within the South Asian group. This finding was incongruent with the mtDNA diversity. Analysis conducted at the individual level showed that a shrew from the central region in Myanmar that carries a South Asian type of mtDNA showed the electrophoretic variants specific to the Southeast Asian group, suggesting that this region is a contact zone between the two major groups.

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

Feasibility of micronucleus methods for monitoring genetic damage in two feral species of small mammals.

Peromyscus leucopus (white-footed mouse) and Cryptotis parva (least shrew) possess desirable attributes for biomonitoring contamination of terrestrial ecosystems, but few studies have examined the potential use of these species for monitoring exposure to genotoxic contaminants. The susceptibility of laboratory-reared C. parva, P. leucopus, and Mus musculus (house mouse, strain CD-1) to micronucleus (MN) induction by known clastogens was evaluated. Animals were exposed for 24 hr to methyl methanesulfonate (MMS; 12.5, 25, and 50 mg/kg), 4-nitroquinoline 1-oxide (4-NQO; 7.5, 15, and 30 mg/kg), or mercuric chloride (HgCl2; 6, 12, and 24 mg/kg). Both MMS and 4-NQO induced dose-related increases in micronucleated polychromatic erythrocytes (MNPCE) in all three species, whereas HgCl2 induced a weak response only in P. leucopus. P. leucopus and C. parva were more sensitive than M. musculus to MMS. Similar micronucleus responses to 4-NQO were seen in each of the species. The feasibility of using blood for MN assessment was evaluated by comparing MN frequencies in bone marrow (BM) PCE, and blood PCE and normochromatic erythrocytes (NCE) in untreated animals, and following daily treatment for 1, 2, 3, and 10 days with 0.4 mg/kg triethylenemelamine (TEM). The results indicated that micronucleated erythrocytes were removed from the circulating blood in P. leucopus, but not in C. parva. Measurement of BM and blood MN levels appears feasible for monitoring exposure to genotoxic agents in C. parva and P. leucopus, and for distinguishing between acute and chronic exposure in C. parva.

Characterization of serum lipoproteins from Suncus: a candidate animal model for abetalipoproteinemia.

We have previously reported that fatty liver was induced in a novel experimental animal, Suncus murinus (suncus), by 24-h fasting and that apolipoprotein B (apo B) was not actively synthesized in the liver. However, a faint signal of apo B mRNA was detected in the liver, suggesting possible synthesis of apo B. Small amounts of VLDL and LDL have been separated from suncus serum by ultracentrifugation. Electron microscopic study of the lipoproteins revealed the existence of small particles in VLDL. High performance liquid chromatographic analysis of the lipoproteins showed that the peaks of TG and cholesterol were mainly at the HDL fraction. These results indicate the existence of lipoproteins as small as HDL which were rich in TG and floated at the density of VLDL upon ultracentrifugation. Apolipoprotein analysis showed two bands of 500- and 200-kDa proteins in VLDL and LDL. Western blot analysis using antibody against the 500-kDa protein revealed reaction not only with suncus 500- and 200-kDa proteins but also with human apo B-100. In conclusion, a small amount of apo B is transported in the suncus serum as VLDL and LDL, although almost all lipid is packed in HDL-size particles.

Differences in levels of erythrocyte glutathione and its metabolizing enzyme activities among primates.

1. The levels of erythrocyte glutathione and the activities of its metabolizing enzymes--glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and glutathione reductase (GR)--were measured in four species of primates: human, rhesus monkey, common marmoset and common tree shrew. 2. There were marked differences in GSH-Px and GST activities among the primates, while GR activity and glutathione level were much less variable.

Alleles at plasma alpha-amylase locus(Amy-1) of the house musk shrew(Suncus murinus): frequency distribution and new variants in laboratory lines and local Asian populations.

Among nine laboratory shrew lines originating from the Japanese islands (Nag, Tok, TKU, Ize, Tr and OKI lines), West Java (Bog), Bangladesh (BAN) and Sri Lanka (SRI), Nag, Tr and Bog were fixed with Amy-1b and SRI with Amy-1a. The remaining lines were still highly polymorphic with the two alleles. A new electrophoretic band C was found in the BAN line and concluded to be expressed by a codominant allele, Amy-1c, which was carried by a single heterozygous female from among eleven wild shrews of the original breeding stock. In most local populations of the Asian shrews surveyed, Amy-1b was more common than Amy-1a. The Sri Lanka population was clearly distinguishable from the others, being nearly fixed with Amy-1a. The C band was found in eleven (one homozygous) of 86 wild shrews caught in Bangladesh. Of a total of 234 wild shrews collected from four Japanese and two Indonesian islands, Bangladesh, and Sri Lanka, only two showed different AMY-bands from AMY-A, -B and -C, and such bands were not found in the laboratory-bred shrews examined.

Serum constituents and liver photomicrographs of wild Suncus murinus in the south of Taiwan.

Serum constituents and liver of the wild male suncus as well as of those bred and fed in our laboratory were examined serologically and histologically. The following results were obtained: The serum glutamic-oxaloacetic transaminase, gamma-glutamyl transaminase, inorganic phosphate, blood urea nitrogen, creatinine, uric acid, and zinc turbidity test levels of wild suncus were higher than those of 4 and 8 week old fed suncus. The total cholesterol level of wild suncus was lower than those of 4 and 8 week old fed suncus. In wild suncus, the total bilirubin and direct bilirubin levels were higher, and the Ca and albumin levels were lower than those of 4 week old fed suncus. However, no significant differences were observed between the wild and the 8 week old fed suncus. Although the alkaline phosphatese and thymol turbidity test levels of wild suncus were larger than those of 8 week old fed suncus, no significant differences were observed between the wild and the 4 week old fed suncus. The histological study revealed the presence of fatty droplets in only 2 of the 67 wild suncus examined while fatty droplets (grade +) were observed in almost all the fed suncus. In one case of the wild suncus, moderate round cell infiltration in interlobular connective tissue was found.

Serum constituents of Suncus murinus.

Blood samples were collected by cardiac puncture from nine-week-old suncus, mice and rats those had been fasted for 16 hours, and the serum was assayed for the levels of corticosterone, cortisol, and other constituents. The following results were obtained: The serum levels of corticosterone and cortisol and corticosterone/cortisol ratio of suncus were about the same as those of human. The SGPT, gamma-GT, ALP, LDH, Ca and K values in suncus were all within the normal range of the respective values in human. SGOT, amylase, BUN, Na, Cl, Fe and inorganic phosphate values were higher, and total cholesterol, triglyceride, phospholipid, creatinine, urea, total protein, albumin and bilirubin levels were lower in suncus than the respective normal values in human. The values of SGOT, amylase, ZTT, and K were higher in female than in male suncus, while the values of gamma-GT and ALP were higher in the male. Feeding the animals individually in the individual cages for a week increased the values of direct bilirubin, total cholesterol, ZTT, Na, K and Cl and lowered the value of urea. The values of SGPT, ALP and triglyceride of suncus obtained in 1984 were higher, the value of albumin was lower, than the respective values obtained in 1983. The value of cholinesterase in suncus was very small.

Genetic polymorphism of the vitamin D binding protein (Gc) in the musk shrew (Suncus murinus).

In the musk shrew (Suncus murinus), the electrophoretic bands in the post-albumin region were identified as vitamin D binding protein (Gc) by the [3H] vitamin D3 binding method. Three Gc phenotypes were distinguished from each other: a single faster band (Gc-A), a single slower band (Gc-B) and the double bands (Gc-AB). Results of mating experiments indicated that the Gc-A and Gc-B are controlled by two codominant alleles, Gca and Gcb at an autosomal locus (Gc), respectively. It was noticed that, in the Gc-AB phenotypes, the Gc-B band was constantly more intense than the Gc-A band in the protein staining. The same tendency was also observed between the homozygous Gc-A and Gc-B bands, and further, radioactivity of the Gc-B bound with [3H] vitamin D3 was about twofold higher than that of the Gc-A. These results suggest that the Gcb yields its protein product twofold more than the Gca. No cross-reaction between the shrew proteins and a rabbit anti-human Gc protein was observed.

The primary structure of adult hemoglobin of musk shrew (Suncus murinus).

An alpha chain and two beta chains beta I and beta II) were obtained from adult hemoglobin of the musk shrew (Suncus murinus) by CM-cellulose column chromatography. The S-carboxymethylated alpha chain and the S-aminoethylated beta chains were each digested with trypsin and the amino acid sequences of the tryptic peptides obtained were established. The ordering of these peptides in the alpha and beta chains was deduced from their homology with the primary structures of the alpha and beta chains of human adult hemoglobin. The sequence of the alpha chain thus determined indicated to be heterogeneous at the 15th position from the N-terminus. On the other hand, comparing the primary structure of beta I chain with that of beta II chain, 4 amino acid exchanges were recognized. Further, the primary structures of the alpha and beta chains of musk shrew hemoglobin were compared with those of alpha and beta chains of human and European hedgehog hemoglobins.

Blood oxygen transport and organ weights of small bats and small non-flying mammals.

Blood oxygen transport properties and organ weights of five bat species in the weight range between 4.8 and 150 g have been investigated and compared to four similar-size non-flying mammals. In addition allometric organ weight-body weight relations have been compiled for both groups. All bats had higher relative heart weights than the terrestrial mammals. In both groups the relative heart weight increased with decreasing body weight. In bats hemoglobin concentrations (18.2--24.4 g Hb/dl), hematocrits (51--63%), and red blood cell counts (11.0 x 10(6)--26.2 x 10(6) RBC/microliter) were remarkably increased compared to non-flying mammals (15.3--17.4 g Hb/dl, 44--50% Hct, 8.8 x 10(6)--18.3 x 10(6) RBC/microliters). The highest values have been found in the smallest european bat Pipistrellus pipistrellus (4.8 g), they exceeded by far the results of the smallest terrestrial mammal, the shrew Suncus etruscus (2.5 g). Oxygen half saturation pressures and Bohr factors did not show considerable differences between similar-size flying and non-flying mammals. Our results indicate that the higher specific oxygen uptake of flying bats compared to exercising non-flying mammals is mainly enabled by larger hearts and larger blood oxygen capacities.