Plant species identification using fecal DNAs from red-eared slider and Reeves’ pond turtle in agricultural canals for rural ecosystem conservation

Fecal DNA samples from the red-eared slider and Reeves’ pond turtle, suspected pests of lotus root paddies, were used to identify the plant species eaten by these turtles in order to develop a strategy for rural ecosystem conservation. The fecal samples were obtained from young and adult individuals (mostly female) of both species living in agricultural canals surrounding lotus root paddies in Tokushima Prefecture, Japan. The samples were screened for the presence or absence of DNA from nine plant species using PCR and plant species-specific primers for the rbcL gene of chloroplast DNA. In the red-eared slider, our analysis identified seven plant species in the fecal DNA samples of adults and three plant species in those of young individuals. In Reeves’ pond turtle, our analysis identified two plant species from adult fecal samples and one species from those of young individuals. Thus, adult red-eared sliders consume a greater range of plants than young red-eared sliders or Reeves’ pond turtles. Both turtle species, independently of age, consumed lotus plants and were likely to cause feeding damage to lotus roots. Considering the plant species detected in adult red-eared sliders and these plant habitats, we suggest that this adult turtle is likely to travel between the agricultural canals and the lotus root paddies. These findings will help the development of strategies for preventing damage to lotus roots by these turtles; furthermore, they indicate that fecal DNA analysis will be applicable to investigation of the feeding habits of other animal species.     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002     

Prognostic significance of surgical margin, Ki-67 and cyclin D1 protein expression in grade II canine cutaneous mast cell tumor

The prognosis for canine cutaneous mast cell tumor (CCMT) is thought to be correlated with histopathological grading. However, the wide variety of histopathologic types of grade II is one of the most troublesome and difficult points for prognosis. The objective of this study is to determine the prognostic value of surgical margin, ki-67 and cyclin D1 protein expression in grade II tumor. Surgically resected specimens of solitary grade II CCMT from 48 dogs with follow-up periods over 360 days (median was 1080 days) were used in this study. The expression of cyclin D1 and ki-67 proteins was determined by morphometrically using slides stained immunocytochemically, and the correlations among the results, survival rate, and recurrence and/or metastasis rate of each dog were analyzed statistically. The recurrence and/or metastasis and mortality rate in the incomplete surgical excision group within 30 months postoperatively were higher than that of the complete surgical excision group. In the incomplete surgical excision group, dogs with low positive staining of ki-67 had a significantly better survival, but the recurrence and metastasis rate and ki-67 positivity failed to show a significant correlation. Only a small number of cases showed cyclin D1-positive tumor cells, but most of them had a poor outcome with a high recurrence rate. In grade II CCMT, incomplete excision induced a relatively high metastasis rate and poor prognosis. Ki-67 positivity is a marker for the estimation of overall survival in incomplete surgical excision cases. Cyclin D1 positivity was low and may not have a prognostic role.     

Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Infection of a chimeric simian and human immunodeficiency virus with CCR5-specific HIV-1 envelope to Rhesus macaques

Human immunodeficiency virus (HIV) infects lymphocytes and macrophages via CD4 and chemokine receptors. In this study, the infectivity of a chimeric simian and human immunodeficiency virus (SHIV) having a CCR5-specific HIV-1 envelope gene was examined. A SHIV strain termed SHIV-JRFL could enter cells via CD4 with a chemokine receptor CCR5, not CXCR4, and the viral replication was suppressed by recombinant human RANTES, one of beta-chemokines. The intravenous inoculation of SHIV-JRFL into two rhesus macaques resulted in a systemic infection, though it was rather weak. During the early infection, the production of RANTES from Con A-stimulated PBMCs of the infected monkeys increased. These results suggested that beta-chemokine has the potential to limit the infectivity of an R5-type SHIV.     

Cerebellar ataxia due to Toxocara infection in Mongolian gerbils,Meriones unguiculatus

We assessed the usefulness of gerbils as an experimental model for neurologic toxocarosis. Mongolian gerbils, Meriones unguiculatus, infected with Toxocara canis or Toxocara cati (1000 eggs/gerbil) showed progressive neurologic disorders from 50 days after infection in T. canis-infected gerbils or from 120 days after infection in T. cati-infected gerbils. The incidence of the onset was 6 of the 13 gerbils (49%) in the T. canis-gerbils and 5 of the 7 gerbils (71%) in the T. cati-gerbils. Histopathologically, the cerebellum was the most affected in both groups. We observed loss of Purkinje cells, glial nerve fibers, and nerve sheaths. We also found foci consisting of aggregated macrophages scattered in the white matter of the cerebellum. The affected gerbils showed ataxia and ultimately died of cachexia. Our findings suggest that irreversible neurologic toxocarosis in gerbils can be induced by infection with either T. canis or T. cati.     

Developmental changes in pituitary–thyroid axis, and formation of gonads in leptocephali and glass eels of Anguilla spp.

SUMMARY: Pituitary, thyroid gland and gonads in leptocephali of Japanese eel Anguilla japonica (19.8–32.6 mm in total length), A. obscura (45.0 mm), and A. bicolor pacifica (49.5 mm) and those in glass eels of the Japanese eel were histologically and immunohistochemically examined in order to observe the developmental changes of these endocrine organs in the Anguillidae. The pituitary, consisting of adenohypophysis and neurohypophysis in Japanese eel leptocephali over 22.5 mm, did not contain thyroid stimulating hormone (TSH) immunoreactive cells. Such cells were, however, detectable in the more developed pituitaries of leptocephali of A. obscura and A. bicolor pacifica and in those of glass eels. Conversely, thyroxine (T₄)-immunoreactive thyroid follicles could be detected in all specimens, both leptocephalic and glass eel. Only in glass eels, gonads were found in the body cavity, and these gonads harbored one or two primordial germ cells (PGC) per cross-section. Our results indicate that thyroid hormones (TH) production started prior to TSH production, and that TSH and TH are both secreted during the metamorphosis from leptocephalus to glass eel. Therefore, it is plausible that the TSH–TH axis is involved in the metamorphosis from leptocephalus to glass eel, but not in the early growth from preleptocephalus to leptocephalus.     

Isolation of Staphylococcus species from the tonsils of healthy cattle and phage patterns of isolates

Staphylococci were found in the tonsils of 121 (75.2%) of 161 cattle. There were 15 different species, 10 belonging to novobiocin-sensitive species. The most predominant species was S. simulans (79.3% of the 121 carriers), followed by S. aureus (20.7%), S. chromogenes (10.7%) and S. epidermidis (8.3%). The other 11 species were present in 0.8 to 5.8%. Twenty-six unidentifiable isolates were isolated from 26 (21.5%) carriers. Sixty-two (51.2%) of the 121 carriers yielded two to five Staphylococcus species together while only one species could be found in each of the other 59 (48.8%). Combinations of S. simulans and other species were most frequently encountered in 50 (41.3%) of the 121 carriers. Twenty-four (96.0%) out of 25 S. aureus isolates, 3 (42.9%) of 7 S. hyicus isolates and 45 (25.4%) of 177 coagulase-negative staphylococci (13 species and unidentifiable isolates) isolates were phage typable. Most of S. aureus isolates were lysed by bovine phages 119 (n = 16) or 116 (n = 5). Thirty-three (25.4%) of 45 coagulase-negative staphylococci typable isolates with Pulverer's phage set showed the phage pattern ph5/ph9/ph10/ph12/ph13/U4/U14/U16/++ +U20/U46. The tonsils of cattle thus appear to be a suitable environment for Staphylococcus species, particularly novobiocin-sensitive species.