Genotyping of Giardia intestinalis from domestic and wild animals in Japan using glutamete dehydrogenase gene sequencing

To determine the genotypes of Giardia intestinalis from domestic and wild animals in Japan, Giardia isolates obtained from feces of 24 dogs kept in households and breeding kennels, three companion cats, five dairy calves and three wild monkeys, Macaca fuscata, were genotyped using the 177 bp sequence of the glutamete dehydrogenase gene (gdh). The genotypes were assemblages A, C, D or A/D for dog isolates, Assemblage F for cat isolates, assemblages A or E for calf isolates and assemblage B for monkey isolates. This is the first report on the genotypes of Giardia isolates from cats, calves and wild monkeys in Japan.     

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.     

Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.     

Analysis of DNA relationships among Aeromonas species by RAPD (randomly amplified polymorphic DNA) typing

Genetic differences between a collection of aeromonads were studied in two laboratories by analysis of randomly amplified polymorphic DNA (RAPD). A single randomly designed primer, generated reproducible profiles of genomic DNA in both laboratories for Aeromonas salmonicida subspecies salmonicida, although the profiles differed between laboratories. Analysis of atypical strains of A. salmonicida and isolates of the A. hydrophila group produced scattered profiles in both laboratories. The uniform fingerprints produced for A. salmonicida subspecies salmonicida indicate genomic homogeneity. The scattered RAPD profiles of the motile aeromonads demonstrate the genomic diversity of this group. A group of unspeciated motile aeromonads gave uniform fingerprints, suggesting the possibility of a genomically homogeneous species. Although the RAPD technique is susceptible to the effects of minor technical variations, this study has demonstrated that where there is DNA similarity, it can be recognized, and where there is diversity, differentiation can be made. RAPD promises to be useful in epidemiological studies for rapid identification of bacteria where a source of reference DNA is available and may be useful in preliminary investigations of relatedness within groups.     

Identification of enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri by expressed sequence tag analysis

Expressed sequence tag (EST) analyses were performed with the aim of identifying enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri. Of the 768 ESTs identified and sequenced, 669 were grouped into 324 clusters. Of these clusters, 123 comprising 245 ESTs were found to be homologous to genes reported to date. Among these, 43 clusters were annotated as enzymes according to the Enzyme Commission (EC) numbering system. Two EC groups, oxidoreductases and hydrolases, possessed a large number of ESTs. A cluster homologous to the glutathione peroxidase, an enzyme in the oxidoreductase group, contained 16 ESTs, which accounted for 2.4% of the total ESTs sequenced. There are three serine proteases, three cathepsins, two triacylgricerol lipases, and two chitinases among the clusters homologous to the enzymes in the hydrolase group. Since the squid liver functions in the digestive process, these enzymes would be involved in food digestion. Our data provide information on the various types of enzymes expressed in the squid liver and may provide a useful basis for further characterization of these enzymes.     

Density and sound-speed contrasts,and target strength of Japanese sandeel <Emphasis Type="Italic">Ammodytes personatus</Emphasis>

Sound-speed and density contrasts (h and g, respectively), important acoustic material properties, of Japanese sandeel Ammodytes personatus were measured to estimate theoretical target strength (TS). The measured sound-speed contrast of adult fish varied between 1.016 and 1.023 (mean 1.020), and showed temperature dependence. The measured density contrast differed significantly between juvenile and adult. The density contrast of juvenile varied between 1.017 and 1.024 (1.021), and that of adult varied between 1.026 and 1.038 (1.032). Using these results, TS at 38 and 120 kHz in the fishing season were estimated by an empirical sound scattering model. TS of an individual fish varied significantly with change of tilt angle. TS of near-dorsal aspect (TS_max) and tilt-averaged TS (TS_ave) differed by up to 7 dB. At both frequencies, two different TS_ave−length relationships (TS_ave = a log L + b) were obtained for adult and juvenile. The coefficients of log L of adult were close to 20, suggesting that backscattering strength was proportional to square of body length. These values were larger in juvenile (34.0 at 120 kHz, 56.5 at 38 kHz), suggesting that backscattering strength varied drastically with the cube or fifth power of body length.     

Algal-Derived Halogenated Sesquiterpenes from Laurencia dendroidea as Lead Compounds in Schistosomiasis Environmental Control

Schistosomiasis has been controlled for more than 40 years with a single drug, praziquantel, and only one molluscicide, niclosamide, raising concern of the possibility of the emergence of resistant strains. However, the molecular targets for both agents are thus far unknown. Consequently, the search for lead compounds from natural sources has been encouraged due to their diverse structure and function. Our search for natural compounds with potential use in schistosomiasis control led to the identification of an algal species, Laurencia dendroidea, whose extracts demonstrated significant activity toward both Schistosoma mansoni parasites and their intermediate host snails Biomphalaria glabrata. In the present study, three seaweed-derived halogenated sesquiterpenes, (−)-elatol, rogiolol, and obtusol are proposed as potential lead compounds for the development of anthelminthic drugs for the treatment of and pesticides for the environmental control of schistosomiasis. The three compounds were screened for their antischistosomal and molluscicidal activities. The screening revealed that rogiolol exhibits significant activity toward the survival of adult worms, and that all three compounds showed activity against S. mansoni cercariae and B. glabrata embryos. Biomonitored fractioning of L. dendroidea extracts indicated elatol as the most active compound toward cercariae larvae and snail embryos.     

Availability of genetically modified feed ingredient: investigations of ingested foreign DNA in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

ABSTRACT:   Foreign DNA fragments from genetically modified defatted soybean meal (GM SBM) in rainbow trout was traced by nested polymerase chain reaction (PCR) and located by in situ hybridization. Either a GM or non-GM SBM formulated diet (42% protein) was fed to fish (average weight 50.5 g) for 2 weeks. The degradation results showed that the cauliflower mosaic virus 35S promoter (220 bp) fragment was detected in the contents of digestive system only in fish fed the GM SBM diet, and it was not detected on the third day after changing the diet to the non-GM SBM diet. For the possible transferal results, the promoter fragment was detected in the leukocyte, head kidney and muscle only of fish fed the GM SBM diet; it was not detected on the fifth day after changing the diet to the non-GM SBM diet. These results suggest that a foreign DNA fragment was not completely degraded and might be taken up into organs through the gastrointestinal tract. However, foreign DNA was not detected after the withdrawal period. Thus, the data show that uptake of DNA from GM SBM might not remain in the tissues of fish fed GM SBM diet.     

Cottony leak of scarlet runner bean caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

Severe cottony leak was found on stems of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture in August 2006. The causal fungus was identified as Pythium aphanidermatum. The name cottony leak of scarlet runner bean (Watagusare-byo in Japanese) is proposed for this new disease.