Evaluation of the water-quality dynamics in a eutrophic agricultural pond by using a one-box ecosystem model considering several algal groups

The Okubo pond is an agricultural pond located in the Itoshima area of Fukuoka Prefecture, Japan. It was constructed for irrigating a nearby cultivated area. A monitoring program from August 11 to November 26, 2008, suggested the risk of eutrophication in the water body. The high total nitrogen concentration (1.34 mg/l), high total phosphorus concentration (0.06 mg/l) and extreme oxygen depletion (2 mg/l on 5 November 2008) exceeded the Japanese standards for paddy irrigation water. Furthermore, luxuriant algal blooming in October indicated a hypereutrophic status according to the OECD (1982) criteria. In this study, a one-box ecosystem model was developed to obtain an insight into the seasonal variations in the algal concentrations and chemical components of the Okubo pond with the aims of protecting its aquatic ecosystem and maintaining its water quality. The model was based on a completely mixed system and included 12 water-quality indices: green algae, blue-green algae, diatoms and dinoflagellates, cryptophytes, zooplankton, particulate organic matter, dissolved organic matter, phosphate–phosphorus, ammonia–nitrogen, nitrite–nitrogen, nitrate–nitrogen, and dissolved oxygen. The monitoring data were used to verify the model simulation. Model evaluation suggested good agreement between the predicted and the observed data for the seasonal variations in the algal, nutrient, and dissolved oxygen concentrations. To determine the sensitivity of the model parameters, a sensitivity analysis was conducted leading to the conclusion that the optimal temperature, growth rate, and respiratory rate of the four algal groups, especially the ideal temperature for blue-green and green algae are the most influential factors determining the variability affecting the model inference. From the model simulation, the water temperature and nutrient concentration were considered to be the key limiting factors controlling alternative algal blooms in this period.     

Development of microsatellite markers in cultivated and wild species of sections <Emphasis Type="Italic">Cepa</Emphasis> and <Emphasis Type="Italic">Phyllodolon</Emphasis> in <Emphasis Type="Italic">Allium</Emphasis>

The potential of microsatellite markers for use in genetic studies has been evaluated in Allium cultivated species (Allium cepa, A. fistulosum) and its allied species (A. altaicum, A. galanthum, A. roylei, A. vavilovii). A total of 77 polymerase chain reaction (PCR) primer pairs were employed, 76 of which amplified a single product or several products in either of the species. The 29 AMS primer pairs derived from A. cepa and 46 microsatellites primer pairs from A. fistulosum revealed a lot of polymorphic amplicons between seven Allium species. Some of the microsatellite markers were effective not only for identifying an intraspecific F₁ hybrid between shallot and bulb onion but also for applying to segregation analyses in its F₂ population. All of the microsatellite markers can be used for interspecific taxonomic analyses among two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Generally, our data support the results obtained from recently performed analyses using molecular and morphological markers. However, the phylogeny of A. roylei, a threatened species with several favorable genes, was still ambiguous due to its different positions in each dendrogram generated from the two primer sets originated from A. cepa and A. fistulosum.     

First report of plum pox virus infecting Japanese apricot (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.) in Japan

For the first time, plum pox virus (PPV) has been detected in commercial Japanese apricot (Prunus mume) trees in Tokyo, Japan. These trees had ringspot or mottle on leaves, color breaking of petals and, occasionally, mild ringspots and malformation on fruits. The virus was identified based on the morphology of virus particles, serology, and RT-PCR. The amplified nucleotide fragment shared 100% identity with a partial coat protein gene of PPV-D isolates.     

Production of anti-virus,viroid plants by genetic manipulations

Many pathogenic plant viruses are RNA viruses, which initiate production of double-stranded RNA intermediates when they replicate in host plant cells. Introduction of double-stranded RNA-specific ribonucleases such as the Schizosaccharomyces pombe derived pac I protein and animal cell derived interferon-induced 2',5'-oligoadenylate synthetase (2-5 Aase)/ribonuclease L (RNase L) system into various plants may make plants resistant to various pathogenic viruses and viroids. We have demonstrated that pac I and 2-5 Aase/RNase L transgenic tobacco plants are resistant to various viruses including tobacco mosaic virus, cucumber mosaic virus and potato virus Y. In addition, pac I transgenic potato plants are resistant to potato spindle tuber viroid. Using Agrobacterium-mediated transformation, we have established a transformation system for chrysanthemum plants and have recently developed pac I transgenic chrysanthemum (Dendranthema grandiflora cv Reagan) resistant to chrysanthemum stunt viroid and have grown them in isolated fields for an evaluation of their effects.     

The role of IFN-gamma in cattle infected with Theirelia sergenti

The spleen plays an important role in cellular immunity leading to parasite elimination, especially in hemo-protozoan parasite infection. In the present study, we quantified the serum IFN-gamma in splenectomized (SP) and non-splenectomized (NSP) cows infected with Theirelia sergenti (TS) to investigate whether the spleen plays a role in the Th1-type cytokine responses in cows following parasite infection. A transient increase in IFN-gamma was observed in the early stages of infection in the NSP cows, and the cows did not develop parasitemia. In contrast, the SP cows showed no IFN-gamma response at the early stage of infection, and the cows developed parasitemia following anemia. The NSP cows produced IgG1 and IgG2 antibodies to TS-specific, whereas the SP cows showed increases only in IgG1. Increased PBMC proliferation by cocanavalin A stimulation was observed concomitant with the IFN-gamma response. Pretreatment with IFN-gamma suppressed the propagation of TS in the SP cattle. These results suggest that the spleen plays an important role in the resistance to TS infection, leading to a Th1-type immune response. IFN-gamma might contribute to the activation of immunocytes and play an important role in the immune response to resist TS proliferation.     

Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan

Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.     

Establishment of a chicken monoclonal antibody panel against mammalian prion protein

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.     

U.S.-type Babesia microti isolated from small wild mammals in Eastern Hokkaido, Japan

Our previous report demonstrated that small wild rodents in Japan harbored two types of novel Babesia microti-like parasites (Kobe and Hobetsu types), but not the type widely distributed throughout the temperate zones of North American and Eurasian Continents (U.S. type). In this study, we surveyed small wild mammals collected at various places in the northern part of Japan, seeking for U.S.-type B. microti. A total of 197 small mammals comprising 10 species, Apodemus speciosus, A. argenteus, Clethrionomys rufocanus, C. rutilus, Eothenomys andersoni, Microtus montebelli, Tamias sibiricus, Sorex unguiculatus, S. caecutiens, and Urotrichus talpoides, were examined. Babesia parasites were detected in A. speciosus, C. rufocanus, C. rutilus, M. montebelli, S. unguiculatus, and S. caecutiens by microscopy of blood smears and by PCR targeting babesial nuclear small-subunit rRNA (rDNA) and beta-tubulin genes. Inoculation of their bloods into experimental animals gave rise to 23 parasite isolates, which included 16 from A. speciosus, 4 from C. rufocanus, and 1 each from C. rutilus, M. montebelli and S. unguiculatus. Sequencing analyses of their rDNA and beta-tubulin genes revealed that, of the 23 isolates, 20 and 3 were of Hobetsu and U.S. types, respectively. The U.S.-type B. microti strains isolated in Japan, however, were distinguishable from the isolates in the United States when their beta-tubulin gene sequences and antigen profiles in Western blots were compared. We conclude that U.S.-type B. microti exists in Japan although it has been genetically and antigenically diversified from that distributed in the United States. The results also suggest that not only rodents, but also some insectivores may serve as reservoirs for the agent of human babesiosis.     

Retrospective seroepidemiological survey for human babesiosis in an area in Japan where a tick-borne disease is endemic

A total of 1,335 archived human sera collected in 1985 from an area in Japan where a tick-borne disease is endemic were examined by indirect immunofluorescence antibody test (IFAT) to estimate seroprevalence against three serologically distinct types of Babesia microti-like parasites; namely, Hobetsu, Kobe, and U.S. types. Eighteen sera (1.3%) were found to be IFAT-positive (titer 1:100-1:6,400), of which 14 and three were ascertained by Western blot analysis to be positive against the Hobetsu and Kobe types, respectively. In addition, four sera showed an IFAT titer of 1:100 against the U.S. type, but they appeared to be false-positive because they were cross-reactive against the Hobetsu and Kobe types, and also because a U.S.-type parasite has not been found in Japan. Our results suggest that human babesiosis in Japan occurred prior to the discovery of the index case in 1999 and that the infections were caused mainly by Hobetsu-type parasites.