Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.     

Effects of hydroxy-aluminum on flocculation and permeability of silt and clay fractions separated from paddy soils

Flocculation tests and permeability measurements were carried out for silt and clay fractions separated from surface and subsurface horizons of four paddy soils. There was not much difference in the flocculating power between A1(OH)_2.2C1_0.8 and CaSO₄. Their flocculation value, 0.3 to 3 mM, was attained by adding the reagents at the rate of 0.24 to 2.4 mmol/100 g of the silt and clay. The flocculation with A1(OH)_2.2C1_0.8 or CaSO₄ did not affect the sediment volume of the silt and clay. This sediment volume was smaller for the surface than for the subsurface horizons of each soil, suggesting that soil organic matter and repetition of wetting and drying contribute to the stable aggregation of the silt and clay in the surface horizon. The permeability was higher for the silt and clay fractions separated from the surface horizon than that from the subsurface horizon, and the addition of 2.5 to 4.2 mmol of A1(OH)_2.2C1_0.8 or CaSO₄/100g of the silt and clay increased its permeability by 1.5 to 4 times. The effect of A1(OH)_2.2C1_0.8 was more lasting than that of CaSO₄ and was recognized even after 200 ml of water passed through 1 g of the silt and clay. The effect of the flocculating reagent on the permeability was more or less enhanced by air-drying the treated silt and clay.     

Rice cultivation and its environmental conditions in the mediterranean countries I. Climate and physiography in relation to rice cultivation

In order to characterize the rice cultivation in the Mediterranean countries, climate and physiography of rice growing areas in Italy, Spain, and Portugal were studied. In northern Italy rice is extensively cultivated on terraces and fluvial plains of the Po and its tributaries, making use of the supply of water from Alpine glaciers in addition to the relatively abundant rainfalls of the area. In the Iberian peninsula summer climate is so dry that rice is cultivable only where the ratio of catchment area to rice land area is sufficiently large, thus rice lands are practically limited to the floodplains and deltas of the big rivers, such as the Sado and the Tejo in Portugal and the Guadalquivir and the Ebro in Spain.

In order to characterize the rice cultivation in the Mediterranean countries, climate and physiography of rice growing areas in Italy, Spain, and Portugal were studied. In northern Italy rice is extensively cultivated on terraces and fluvial plains of the Po and its tributaries, making use of the supply of water from Alpine glaciers in addition to the relatively abundant rainfalls of the area. In the Iberian peninsula summer climate is so dry that rice is cultivable only where the ratio of catchment area to rice land area is sufficiently large, thus rice lands are practically limited to the floodplains and deltas of the big rivers, such as the Sado and the Tejo in Portugal and the Guadalquivir and the Ebro in Spain.     

Evaluation of the efficacy of Salmonella enteritidis oil-emulsion bacterin in an intravaginal challenge model in hens.

The efficacy of Salmonella enteritidis (SE) oil-emulsion bacterin (a commercially available vaccine) was evaluated in an intravaginal challenge model in hens producing a high rate of SE-contaminated eggs. Hens were vaccinated at 38 wk of age. A second (booster) bacterin injection was administered 4 wk later. Two weeks after the second vaccination, all hens were challenged intravaginally with 10(7) colony-forming units of SE. After challenge, 36 of 189 eggs (19.0%) in the vaccinated hens were positive for SE, and this contamination rate was significantly (P < 0.01) lower than that in the unvaccinated hens (61 of 165 eggs, 37.0%). SE was highly recovered from the cloacal and vaginal swabs of the unvaccinated and vaccinated hens, but the number of SE from the cloaca of the vaccinated hens was significantly (P < 0.05) lower than that in the unvaccinated hens at 7 days post-challenge (PC). The recoveries of SE from the spleen and ovary in the vaccinated hens were significantly (P < 0.05) lower than those in the unvaccinated hens at 7 days PC. At necropsy, SE was recovered from 2 of 15 forming eggs (13.3%) taken from the oviducts of the unvaccinated hens, whereas no SE was recovered from 17 forming eggs in the vaccinated hens. After vaccination, serum antibodies for SE in the vaccinated hens were significantly higher than those in the unvaccinated hens. Antibodies from the oviductal washing, especially immunoglobulin G isotype, in the vaccinated hens were higher than those in the unvaccinated hens after challenge. This intravaginal challenge model produced frequent contaminated eggs and clearly demonstrated the ability of the bacterin to protect against egg contamination. The present model may be a useful tool for further studies to evaluate the protective effect against SE contamination of eggs by potential vaccine candidates.