Effect of increased feeding of dietary α‐linolenic acid by grazing on formation of the cis9,trans11–18:2 isoform of conjugated linoleic acid in bovine milk

Feeding systems such as grazing affect the fatty acid profile of bovine milk fat. In addition, milk fat is formed as the product of fatty acid metabolism in cow bodies before being secreted into milk. However, how grazing influences milk fatty acid profile through the metabolism has not been completely characterized. When fatty acid concentrations in Holstein milk were compared between grazing and non‐grazing periods, α‐linolenic acid was significantly higher in the grazing period than in the non‐grazing period. This could be explained with an increase in α‐linolenic acid feeding with grazing. α‐linolenic acid had a linear positive correlation with conjugated linoleic acid (9c,11t‐18:2) (CLA) and vaccenic acid (VA) during the grazing period, whereas CLA had higher correlation with linoleic acid rather than with α‐linolenic acid during the non‐grazing period. These data indicate that the high content of dietary α‐linolenic acid affects CLA and VA formation in milk of grazing periods via α‐linolenic acid metabolism into VA.     

A 90-day Feeding Toxicity Study of l-Serine in Male and Female Fischer 344 Rats

A subchronic feeding study of l-serine (l-Ser) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0, 0.06, 0.5, 1.5 or 5.0% concentrations of l-Ser for 90 days. There were no toxicologically significant, treatment-related changes with regards to body weight, food intake, water intake or urinalysis data. In several of the hematology, serum biochemistry and organ weight parameters, significant changes were observed between some of the treated groups and the controls. All these changes, however, were subtle and lacked any corresponding pathological findings. In addition, the increased or decreased values remained within the range of the historical control values. In fact, histopathological assessment revealed only sporadic and/or spontaneous lesions. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Ser was, therefore, determined to be at least a dietary dose of 5.0% (2765.0 mg/kg body weight/day for males and 2905.1 mg/kg body weight/day for females) under the present experimental conditions.     

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.