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We investigated the effect of rice grain conservation methods on feed intake, milk production, blood metabolites, and rumen fermentation in dairy cows. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS-A), or ensiled before crushing (ENS-B). Twelve multiparous Holstein cows were used in a replicated 3 × 3 Latin square design with three dietary treatments comprising ad libitum access to one of three total mixed rations (TMRs; containing DRY, ENS-A, or ENS-B at 17% of dietary dry matter) plus a standard allowance of 2.0 kg/day of dairy concentrates. The dietary treatments did not affect the feed intake, milk yield, or milk composition. The selected blood constituents were not influenced by the rice conservation method. The ruminal lactic acid and total volatile fatty acid (VFA) concentrations and the VFA proportion in the cows were not influenced by the rice conservation method. These results demonstrate that the rice grain conservation method has little impact on lactation performance when cows are fed a TMR containing 17% treated rice grain (dry matter basis).

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells–large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4μ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1–induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.

We evaluated the effects of different methods of conserving the total mixed ration (TMR) and processing hulled rice (Oryza sativa L.) on intake, digestion, ruminal fermentation, lactation performance, and nitrogen utilization in dairy cows. Eight multiparous Holstein cows (126 ± 19 d in milk) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of dietary treatments. The experimental diets used fresh TMR and ensiled TMR containing either dry-rolled (DR) hulled rice or steam-flaked (SF) hulled rice. The fresh TMR was prepared every morning. The ensiled TMR was prepared by baling fresh TMR and then sealing it with a bale wrapper; this was stored outdoors at 10 to 30°C for >4 mo. The method of grain processing did not affect the dry matter (DM) intake. The DM intake tended to be greater for ensiled TMR than for fresh TMR. Apparent total-tract DM digestibility and milk yield were increased by feeding ensiled TMR instead of fresh TMR and by replacing DR with SF. An interaction effect between the TMR conservation method and the grain processing method was detected for DM digestibility and milk yield; replacing DR with SF increased the DM digestibility and milk yield in cows fed fresh TMR, but this did not affect the cows fed ensiled TMR. The milk fat and lactose contents did not differ among dietary treatments. The milk protein concentration was higher for the cows fed SF processed hulled rice than those fed DR, but it was not influenced by the TMR conservation method. The ruminal total volatile fatty acid concentration was higher for the cows fed ensiled TMR compared with those fed fresh TMR, but it was not affected by the grain processing method. The molar proportion of acetate was decreased and propionate was increased by feeding ensiled TMR instead of fresh TMR and by replacing DR with SF. The concentrations of rumen ammonia N and plasma urea N were higher for the cows fed ensiled TMR than fresh TMR and were lower for SF than DR. Feeding ensiled TMR instead of fresh TMR increased the cows' urinary N excretion and decreased the retention N. Replacing DR with SF decreased the urinary N excretion, increased the milk N secretion, and then improved the nitrogen efficiency. These results show that feeding ensiled TMR instead of fresh TMR has an adverse effect on N utilization, but it increases digestion and milk production. Replacing DR with SF also increases digestion, milk yield, and milk protein content, and the improvement of milk yield by replacing DR with SF was prominent in the cows fed fresh TMR.

Blood α-tocopherol (α-Toc) concentrations decline gradually throughout the prepartum period, reaching the nadir after calving in dairy cows. The 6 α-Toc-related molecules [α-Toc transfer protein (TTPA); afamin; scavenger receptor class B, Type I; ATP-binding cassette transporter A1; tocopherol-associated protein (SEC14L2); and cytochrome P450 family 4, subfamily F, polypeptide 2 (CYP4F2)] are expressed in liver and other peripheral tissues. These molecules could regulate α-Toc transport, blood concentrations, and metabolism of α-Toc. Therefore, the aim of this study was to evaluate the changes in the expression of α-Toc-related genes in liver and mammary gland tissues of dairy cows around calving, which have remained elusive until now. In experiment (Exp.) 1, 28 multiparous Holstein cows were used (from -5 to 6 wk relative to parturition) to monitor the changes in dietary α-Toc intake, blood concentrations of α-Toc, and lipoproteins; in Exp. 2, 7 peripartum Holstein cows were used (from -4 to 4 wk relative to parturition) for liver tissue biopsy; and in Exp. 3, 10 peripartum Holstein cows were used (from -8 to 6 wk relative to parturition) to carry out the mammary gland tissue biopsy and milk sampling. In Exp. 1, the serum α-Toc concentrations declined gradually with decreasing amount of α-Toc intake and plasma high-density lipoprotein concentrations toward calving time. However, in the early lactation period after calving, serum α-Toc concentrations remained at a lower concentration despite the recovery of α-Toc intake and plasma high-density lipoprotein concentrations. In Exp. 2, just after calving, the TTPA, SEC14L2, afamin, and albumin mRNA expression levels in the liver were temporarily downregulated, and the hepatic mRNA levels of endoplasmic reticulum stress-induced unfolded protein response markers and acute-phase response marker increased at calving. In Exp. 3, the concentrations of α-Toc in colostrum were greater than those in precolostrum (samples were collected at wk -1 relative to parturition) and mature milk. The expression of TTPA, SEC14L2, and CYP4F2 mRNA in bovine mammary gland tissue was detected. However, TTPA and SEC14L2 mRNA expressions showed the opposite trends: the expression levels of TTPA mRNA peaked whereas SEC14L2 mRNA reached a nadir at calving. These results indicate that the expression of α-Toc-related genes involved in specific α-Toc transfer and metabolism in the liver and mammary gland are altered during calving. Moreover, these changes might be associated with the maintenance of lower serum α-Toc concentrations after calving.

We investigated the effects of different rice conservation techniques on in situ ruminal degradation and in vivo nutrient digestibility and rumen fermentation in steers. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS-A), or ensiled before crushing (ENS-B). Six ruminally cannulated steers were used in a replicated 3 × 3 Latin square design with three dietary treatments: diets containing DRY, ENS-A, or ENS-B at 36% of the dietary dry matter. The in situ rapidly degradable fraction and effective ruminal degradability were higher for ensiled rice than for DRY, and higher for ENS-A than for ENS-B. The ruminal pH was lower and the lactic acid and total volatile acid concentrations were higher for the steers fed ensiled rice than those fed the DRY diet, but a treatment effect was not observed in the comparison between ENS-A and ENS-B. The whole-tract digestibility of crude protein and ether extract was improved when the rice grain was ensiled, but there were no differences in nutrient digestibility between ensiling methods. These results show that ensiling treatment can be a strategy to improve the nutrient value of rice grain, but the ensiling method has little impact on in vivo digestion.

The unfolded protein response (UPR) describes a process involved in the homeostasis of endoplasmic reticulum (ER) and the differentiation of secretory cells. At present, the roles of UPR in the mammary gland tissue of dairy cattle are unknown. In the current study, we investigated the expression of UPR-related genes in Holstein cows during the developmental and lactating stages of the mammary gland tissue. To investigate the roles of UPR during the differentiation of mammary epithelial cells (MEC), we used MAC-T cells, a line of MEC. We collected samples of mammary gland tissue in dairy cows by biopsy during the late gestation and lactation periods and examined the expression of UPR-related genes by quantitative real-time PCR. Expression levels of the spliced X-box binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) were found to be significantly higher in the mammary gland tissue 10 d before delivery compared with 40 d before delivery. An investigation before and after differentiation in MAC-T cells showed that the expression of ATF4 increased after differentiation of MEC, whereas that of the spliced XBP1 did not significantly change. Western blot analysis revealed that the differentiation-inducing stimulus induced phosphorylation of eukaryotic initiation factor 2α (eIF2α) but reduced that of protein kinase RNA-like endoplasmic reticulum kinase (PERK). Additionally, in ATF4-knockdown bovine MEC, differentiation was significantly suppressed; ATF4 knockdown also significantly suppressed the expression of glucocorticoid and insulin receptors. These results revealed that ER stress-independent ATF4 is involved in the cell differentiation mechanism, either directly or indirectly, via the control of the expression of lactogenic hormone receptors in bovine MEC. Immediately after parturition, gene expression levels of the spliced XBP1, ATF4, and C/EBP homologous protein (CHOP) markedly increased in mammary gland tissue, with a strong negative correlation between expression of CHOP and initial milk yield; CHOP is an apoptosis-related protein induced by ER stress. The above findings indicate that UPR is intrinsically associated with apoptosis of MEC, thus affecting the differentiation of these cells, as well as milk yield.

We evaluated the effects of different methods for conserving and crushing rice grain on rumen fermentation characteristics and whole-tract apparent digestibility in steers. The preparation of dietary treatments followed a 2 × 2 factorial design with conservation method (drying vs . ensiling) and crushing method (using an impeller crusher vs . a double–roller crusher) of rice grain as main effects. Rice grain was crushed by the impeller crusher (volume weight: 490.4 g/L, mean particle size: 1382 μm) or the double–roller crusher (volume weight: 373.9 g/L, mean particle size: 2043 μm). Dried rice was prepared by drying to adjust the moisture to 100 g kg −1 . Ensiled rice was prepared by sealing crushed rice with the addition of water and lactic acid bacteria to adjust the moisture to 300 g kg −1 , and stored for 4 months. Eight ruminally cannulated steers (366 ± 32.2 kg initial body weight [BW]) were randomly assigned to a replicated 4 × 4 Latin square design. The experimental diets contained 434 g kg −1 grass silage, 96 g kg −1 soybean meal and 470 g kg −1 treated rice grain (dry matter [DM] basis). Steers were offered experimental diets at 1.55 kg DM/100 kg of BW/day. The conservation method had a large impact on the ruminal fermentation and nutrient digestibility. The ruminal fermentation characteristics and nutrient digestibility (except for starch digestibility) were not affected by the crushing method. No significant interactions were observed between the conservation and crushing methods of rice grain for all fermentation and digestion measurements. The ruminal pH was lower ( P 0.01) and the lactic acid and total volatile fatty acid concentrations were higher ( P 0.01 and P = 0.02, respectively) for the steers fed ensiled rice than those fed dried rice. The proportions of acetate, propionate and butyrate were not affected by ensiling treatment. The whole-tract apparent digestibility of organic matter, crude protein and starch were increased ( P = 0.04, P 0.01 and P 0.01, respectively) and the digestibility of neutral detergent fiber and acid detergent fiber were decreased ( P = 0.046 and P = 0.03, respectively) by replacing dried rice with ensiled rice. The starch digestibility was higher ( P 0.01) for steers fed rice crushed by the impeller crusher compared to the double-roller crusher. These results show that the feeding of ensiled instead of dried rice increases the likelihood of decreasing fiber digestibility because it reduces ruminal pH, but it improves organic matter, crude protein and starch digestion, and thus an ensiling process can be a strategy to improve the nutrient value of rice grain.

We evaluated the effects of replacement of heading stage harvested timothy silage with early-harvested orchardgrass-perennial ryegrass mixed (OP) silage while maintaining or reducing concentrate input on dry matter intake (DMI), milk production, nutrient digestibility, and N balance in dairy cows. Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design with three dietary treatments: TYL, a diet containing timothy silage where forage-to-concentrate ratio (FC) was 50:50; OPL, a diet containing OP silage where FC ratio was 50:50; and OPH, a diet containing OP silage where FC ratio was 60:40. We observed that an equal replacement of timothy with OP silage increased DMI, milk yield, milk protein production, and nutrient digestibility but decreased milk fat content (TYL versus OPL). We observed that replacing timothy with OP silage while reducing concentrate input increased milk fat and protein yield, nutrient digestibility, and feed efficiency and reduced urinary N loss with no effect on DMI or milk fat content (TYL versus OPH). These results show that replacing timothy with OP silage can be a good approach to improve milk production, feed efficiency, and N utilization and reduce concentrate input. However, milk fat depression should be considered when an equal substitution is performed.

The objective of this study was to evaluate the effect of replacing corn with brown rice (BR) on dry matter intake (DMI), milk production, blood metabolites and energy balance (EB) in cows during the periparturient period. Twenty-four nonlactating multiparous Holstein cows in late gestation were studied from 3 week before the expected calving date through 6 week after parturition. The cows were divided into one of two dietary treatment groups based on parity and the expected calving date. The treatments were a diet containing steam-flaked corn and a diet containing steam-flaked BR. Prepartum DMI and body weight (BW) and postpartum DMI, BW, milk yield, milk protein and lactose yield were not affected by the dietary treatment. However, the milk fat content and fat yield were higher ( P = 0.02 and P = 0.01) for cows fed BR than for cows fed corn. Thus, cows fed BR-based total mixed ration (TMR) tended to have more negative EB in early lactation ( P = 0.06) compared with cows fed corn-based TMR. During the prepartum period, the plasma concentrations of glucose, insulin, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), aspartate transaminase (AST) and Ca were not affected by dietary treatments. On the other hand, the postpartum plasma glucose and insulin concentrations were decreased ( P P = 0.02) and the BHBA and AST concentrations were increased ( P P = 0.02) by replacing corn with BR with no dietary effect on postpartum plasma NEFA and Ca concentrations. These results show that feeding BR instead of corn does not affect DMI or milk yield, but increases milk fat production and leads to a more negative EB state in early lactation. Replacing corn with BR during the periparturient period has adverse effects on the metabolism and energy status in early lactating cows.

The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain on feed intake, lactation performance, ruminal fermentation, digestion, and N utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk) were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn (dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7% alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo. Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed 40% corn. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The ruminal ammonia-N concentration decreased as the percentage of BR in the diets was elevated. The proportion of acetate decreased, and that of propionate and butyrate increased with the increasing levels of BR. Plasma urea-N concentrations was lower and glucose and insulin concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The whole-tract apparent digestibility of dry matter, organic matter, and starch increased, and the digestibility of neutral detergent fiber and acid detergent fiber decreased with the increasing BR level in the diet, with no dietary effect on crude protein digestion. As a proportion of N intake, the urinary N excretion was lower and the retention of N was higher for cows fed 40% BR than for cows fed 40% corn, with no dietary effect observed on N secretion in milk and fecal N excretion. These results show that substituting BR for corn decreases urinary N losses and improves N utilization, but causes adverse effects on milk production when cows are fed high-grain diets at 40% of dietary dry matter.

Some anthocyanins have been reported to exhibit antioxidant activity and to enhance antioxidant enzyme activity. The aim of this study was to determine the fermentative quality of purple rice, which is abundant in anthocyanin, and its effects on digestibility, ruminal fermentation and oxidative status markers in sheep. The whole crop silages of purple and control rice were prepared in drum silos after the materials were chopped and lactic acid bacteria was inoculated. These were followed by a feeding trial using eight wethers in a 2 × 2 crossover design. Both the purple and control rice silages showed excellent fermentative qualities. Although the purple rice silage contained abundant anthocyanin, it had inadequate nutritional values in comparison to the control rice silage, including higher levels of fiber fractions, lower nutrient digestibility and lower production of ruminal volatile fatty acids. The feeding of the purple rice silages led to an elevation in plasma activity of superoxide dismutase, which is an important antioxidant enzyme. The results of this study suggest that purple rice may become a source of anthocyanin to improve oxidative status in ruminants. To achieve that, however, a new variety of purple rice with good nutritional value is needed for practical use as a ruminant feed.

Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design to determine the effects of substituting corn grain with brown rice (BR) grain in total mixed ration (TMR) silage on milk yield, ruminal fermentation and nitrogen (N) balance. The TMR silages were made from the ensiling of TMR containing (dry matter basis) 50.1% forage in rice silage and corn silage combination, and 49.9% concentrate. The grain portion of the diets contained 31.2% steam-flaked corn, 31.2% steam-flaked BR or an equal mixture of corn and BR. Dietary treatments did not affect dry matter intake, milk yield and milk fat, protein and lactose yields. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The urinary N excretion decreased linearly (P < 0.01) in response to increased levels of BR, with no dietary effect on N intake, N secretion in milk and fecal N excretion. Our results indicate that steam-flaked BR is a suitable replacement for steam-flaked corn in dairy cow diets, and that it can be included in rations to a level of at least 31.2% of dry matter without adverse effects on milk production, when cows were fed rice silage and corn silage-based diets.

The objective of this experiment was to investigate the effect of hay quality and form on mean retention time (MRT) of digesta and total tract apparent digestibility (TTAD) in horses fed under ad libitum or restricted conditions using four Hokkaido native horses. Two experiments (Exp.) were conducted using 4 × 4 Latin square designs with treatments arranged factorially with hay quality [high quality timothy hay harvested at late vegetative-stage (HH) or low quality timothy hay harvested at late bloom-stage (LH)] and physical form [chopped to a theoretical 10-mm length (G10H) or ground through a 5-mm sieve (G5H)] as main effects. In Exp. 1, horses were offered the experimental diets ad libitum whereas in Exp. 2 horses were offered a restricted allowance of 1.6 kg dry matter (DM) per 100 kg of BW per day. Under ad libitum conditions, hay quality, physical form and their interactions did not affect feed intake. In both Exp., solid phase digesta MRT in total tract and in the first compartment which having the longest MRT were longer (Exp. 1: P=0.08 and P=0.02, Exp. 2: P=0.04 and P=0.04, respectively) for G5H than for G10H, but hay form did not affect MRT in the second compartment which having the second longest MRT. Hay quality did not affect MRT. The MRT through the gastro-intestinal tract of the solid phase was 23.7 h in Exp. 1 and 27.1 h in Exp. 2. The TTAD of DM was higher (P

自由採食下での泌乳初期初産牛の反芻胃容積，消化管部位の内容量，発酵様相，内容物滞留時間を調べた．ホルスタイン種初産牛6頭に分娩後37日から牧草サイレージ，トウモロコシサイレージ，大豆粕，配合飼料からなる混合飼料を自由採食させ，分娩後47日に屠殺し，消化管を10部位に分割し，各組織と内容物を採取した．注水法で測定した反芻胃容積は131.9 Lで，反芻胃容積に対する内容物量は，総量，乾物量，液量でそれぞれ511.1，84.1，427.0 g/Lであった．総VFA濃度は，反芻胃で148.9 mmol/L, 後腸前部位（盲腸から円盤結腸）で84.6-87.3 mmol/L, 後腸後部位（遠位結腸以降）は73.1-74.2 mmol/Lであった．滞留時間は，反芻胃が26.0時間で全消化管（34.1時間）の76.1％を占めたのに対し，第三胃および後腸部位（盲腸以降）では2.7（7.9％）および3.3時間（9.8％）であった．以上の結果から，泌乳初期の初産牛の反芻胃内容量は，泌乳前期以降あるいは泌乳初期の経産牛のそれと比べ少ないこと，また後腸内の内容物滞留時間は短く，泌乳初期牛においては後腸内発酵の寄与が小さいことが示唆された．

初産牛における泌乳初期の採食量と採食行動の関係を明らかにするため，ホルスタイン種初産牛6頭（P），経産牛6頭（M, 平均産次 : 2.2）を用い，分娩後10-16日（＋2週），24-30日（＋4週）における採食量，採食行動，消化管内滞留時間（MRT）を測定した．供試牛には牧草サイレージ，トウモロコシサイレージ，大豆粕，配合飼料からなる混合飼料とイネ科乾草を自由採食させた．採食量は，PがMより，また＋2週が＋4週より少なかった．ミールサイズは，PがMより，また＋2週が＋4週より小さかった．P，Mともに＋2週が＋4週よりミール継続時間は短く，回数は多かった．固相の第一胃MRTは，Pでは分娩後時期で差はないが，Mで＋4週が＋2週より短かった．以上の結果から，P，Mとも分娩後日数の経過に伴い継続時間が長く，サイズの大きいミールが少数回出現する採食パターンに変化し，採食量が増加することが示唆された．またこの変化は，MではMRTが短縮するため，PではMRTは短縮しないものの消化管容積が拡大し，内容物量が増加するためと推察された．

妊娠末期の乳牛における採食量と採食行動の関係を明らかにするため，分娩前37-31日（－5週），23-17日（－3週）および9-3日（－1週）の自由採食量，採食行動および消化管滞留時間を測定した．ホルスタイン種乳牛10頭を2群に分け，分娩予定日の45日前から分娩日までイネ科牧草サイレージ（GS）主体飼料（GS区）あるいはGS区のGSの25％をトウモロコシサイレージに代替した飼料（CS区）を自由採食させた．いずれの項目においても給与飼料による差はなかった．自由採食量は分娩3日前まではほぼ一定で推移した（13.4 kgDM/日）．採食期（ミール）回数は－1週で最も多く（P ＜ 0.05），この時ミールサイズは小さくなる傾向であった（P ＜ 0.1）．また，1ミールあたりの平均継続時間は，－1週が－5週より短かった（P ＜ 0.05）．1日の反芻時間，反芻期数および継続時間は分娩前時期による影響はなかった．第一胃および全消化管内滞留時間は，－1週が－5週に比べ短かった（P ＜ 0.05）．

A recent case of canine histoplasmosis, the first confirmed case of disseminated infection accompanied by carcinoma in Japan, was diagnosed by clinical characteristics, histopathological examination, chest radiographs, ocular fundoscopy and molecular biological data. The clinical manifestations were not limited to cutaneous symptoms but were referable to disseminated infection, similar to human autochthonous cases. The partial sequences of the internal transcribed spacer (ITS1/2) regions of the ribosomal DNA genes of this and other Japanese canine histoplasmosis strains were 99-100% identical to the sequence AB211551 derived from a human isolate in Thailand, and showed a close relationship to the sequences derived from Japanese autochthonous systemic and cutaneous human cases. The phylogenetic analysis of 97 sequences of the ITS1/2 region disclosed six genotypes. The genotypes derived from Japanese autochthonous human and dog cases belonged to the cluster consisting of Histoplasma capsulatum var. capsulatum and H. capsulatum var. farciminosum sequences, indicating that these varieties might cause not only cutaneous but also systemic histoplasmosis, regardless of their host species. The current status of the 3 varieties of Histoplasma capsulatum according to the host species remains a subject of further investigation.

This study investigated the prevalence of C. dubliniensis in a Brazilian family with an HIV - infected child. A total of 42 oral isolates were obtained from eight family members. The identification of C. dubliniensis was performed by polymerase chain reactions (PCR) using primers against a specific sequence of the C. dubliniensis cytochrome b gene. Only the HIV-infected child and his grandmother were colonized by C. dubliniensis. In this study C. dubliniensis isolated from the HIV-infected child exhibited high susceptibility for azoles tested with MICs of 0.125 and 0.5 µg/mL for voriconazole and fluconazole, respectively. Accumulation of [³H] fluconazole in C. dubliniensis isolated from the HIV-infected child was slightly reduced in comparison to the reference susceptible strain. C. dubliniensis isolates had significantly lower ergosterol levels in comparison to C. albicans reference strains.O presente estudo investigou a prevalência de C. dubliniensis em uma família brasileira com uma criança infectada pelo vírus HIV. Um total de 42 isolados orais foram obtidos de 8 membros da família. A identificação de C. dubliniensis foi realizada por polymerase chain reactions (PCR) usando primers contra a sequência específica para o gene C. dubliniensis cytochrome b. Apenas a criança infectada pelo vírus HIV e a avó estavam colonizados por C. dubliniensis. Neste estudo C. dubliniensis isolado da criança infectada pelo vírus HIV exibiu alta susceptibilidade para azoles com concentração mínima inibitória de 0.125 and 0.5 µg/mL para voriconazole and fluconazole respectivamente. Acúmulo de [³H] fluconazol intra-celular foi ligeiramente reduzido em C. dubliniensis isolado da criança infectada pelo vírus HIV em comparação com a cepa referência sensível ao fluconazole. Isolados de C. dubliniensis neste estudo apresentaram níveis significantemente reduzidos de ergosterol da membrane celular em comparação com C. albicans.

Declining incidence of oropharyngeal candidosis and opportunistic infections over recent years can be attributed to the use of highly active anti-retroviral therapy (HAART). Infection with C. albicans generally involves adherence and colonization of superficial tissues. During this process, budding yeasts are able to transform to hyphae and penetrate into the deep tissue. Using the biocell tracer system, C. albicans hyphal growth was dynamically observed at the cellular level. Ritonavir was effective in the inhibition of hyphal growth with growth rate of 0.8 mum/min. This study showed the in vitro effect of HIV anti-retroviral drug on the growth rate of the C. albicans hyphae.O declínio na incidência de candidose orofaríngea e infecções oportunistas associadas a infecção pelo HIV tem sido atribuído a introdução da terapia antiretroviral combinada (HAART). Infecção por C. albicans envolve aderência e colonização da mucosa superficial. Durante este processo leveduras são capazes de transformar-se na forma de hifas e penetrar nos tecidos mais profundos. Usando o sistema "Bio-Cell Tracer", o crescimento de hifas de C. albicans foi observado dinamicamente a nível celular. Ritonavir, inibidor de protease do HIV, foi efetivo na inibição do crescimento de hifas com media de 0.8 mim/min.O presente estudo demonstrou o efeito in vitro de um agente anti-retroviral HIV sobre o crescimento de hifas de C. albicans.

The Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University is the only organization in Japan to possess a series isolates of Coccidioides spp., which are the most virulent pathogenic fungi and which are treated as biosafety level 3 microorganisms. Recently, the genus Coccidioides has been classified into two species, C. immitis and C. posadasii, based on their endemic areas and genotyping; the former species is endemic to the state of California, and the latter is endemic to other parts of North and South America. We reevaluated 19 isolates of Coccidioides immitis stored in our center using a multiple gene analysis. Five isolates were identified as C. immitis and 14 as C. posadasii. Their sequence information in GenBank will help to identify the two genospecies of Coccidioides spp.

Fecal bacteria and protozoa of Hokkaido native horses and light horses were enumerated to compare seasonal variation in hindgut microbes and fermentation between the two breeds. Fecal samples were collected in winter and summer from eight horses (four for each breed) that had been reared together under the same conditions after birth (on woodland pasture in winter and on grassland pasture for the rest of the year). Total fecal bacteria counts for both breeds showed temporal variation, with the highest levels occurring in summer (P

In this study, Swiss mice were experimentally infected with Paracoccidoides brasiliensis (Pb18) and we investigated the levels of gp43 in urine and plasma, anti-gp43 and IgG-gp43 immune complexes in plasma. These levels were correlated with the histopathological findings. Blood and urine samples were collected from mice at 7, 28, 56 and 84 days after intravenous inoculation of 10(5) yeast cells, and analysed by ELISA. The results showed increased levels of soluble gp43 in the plasma in all periods, and anti-gp43 IgG and immune complexes after day 28. High gp43 levels were detected in the urine, except for day 28, coincident with the presence of compact granulomas in lungs. All the infected mice showed fungal cells in the lungs, with initial granulomatous lesions at day 7, dissemination of lesions to other organs at day 56, and granulomas lacking the surrounding mononuclear cells infiltration, especially at days 56 and 84. Our results suggest that gp43 diffuses passively into the urine, and the determination of gp43 levels in urine samples may be a non-invasive alternative method for diagnosis and follow up of PCM. Further studies are needed to determine if the cellular immune response correlate with decreased urine gp43 levels.

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

北海道和種馬に同一原料牧草（チモシー）から調製した無細切の乾草およびサイレージを乾物で同量（2.2％BW）給与し，消化率，咀嚼活動，糞の粒度分布および消化管内容物の平均滞留時間を比較検討した．乾物および繊維成分の消化率には飼料間で差はなかった．乾物摂取量あたりの咀嚼回数は乾草よりサイレージが多かったが（4.6 vs. 5.3回/gDM, P＜0.05），糞の粒度分布は乾草摂取時よりもサイレージ摂取時が大粒子画分（2.36mm以上）の割合が高く（29 vs. 51％, P＜0.05），小粒子画分（0.30mm以上1.18mm未満）の割合が低かった（35 vs. 23％, P＜0.05）．消化管内容物の平均滞留時間は，乾草よりもサイレージが，液相（21.1 vs. 16.9h, P＜0.01）および飼料全体（23.7 vs. 21.8h, P＜0.05）ともに短く，また両飼料とも液相よりも飼料全体が長かった（P＜0.01）．葉部および茎部の滞留時間には両飼料とも差がなかった．

The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods. MICs of micafungin, amphotericin B, itraconazole, and fluconazole for Histoplasma capsulatum var. capsulatum , Blastomyces dermatitidis , Paracoccidioides brasiliensis , Penicillium marneffei , and Sporothrix schenckii were determined both for the yeast-like form and mycelial form. Coccidioides immitis was tested only in its mycelial form. We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi. Micafungin exhibited potent activity against the mycelial forms of H. capsulatum , B. dermatitidis , and C. immitis (MIC range, 0.0078 to 0.0625 μg/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 μg/ml). Micafungin was also more active against the mycelial forms than the yeast-like forms of Paracoccidioides brasiliensis , Penicillium marneffei , and S. schenckii . The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of B. dermatitidis and Paracoccidioides brasiliensis . There was no apparent difference in the activity of itraconazole between the two forms. The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for B. dermatitidis . These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results. At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome. However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.

An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from Fonsecaea species was designed. This set yielded an amplicon with 333 bp for all strains of Fonsecaea pedrosoi and Fonsecaea compacta examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus Fonsecaea .

Histoplasmosis is a fungal infection caused by Histoplasma capsulatum and is distributed a worldwide. Although the disease has been treated as an imported mycosis, some autochthonous human, 1 equine and 4 canine cases suggested that the disease is endemic. Histoplasmosis is classified depending on the variety of causative agent. Histoplasmosis farciminosi known as pseudofarcy, is manifested only in Perissodactyla where it invades lymph nodes and lymph ducts, and is recognized by isolation from horses. Historically, Japan was one of the endemic areas of pseudofarcy before World War II, and more than 20,000 cases were recorded in horses used by the military. Interestingly, Japanese canine histoplasmosis uniformly showed skin ulcers and granulomatous lesions on the skin without pulmonary or gastrointestinal involvement, both of which were very similar to pseudofarcy. It was diagnosed as histoplasmosis by the detection of internal transcribed spacer legions of rRNA gene of H. capsulatum from paraffin embedded tissue samples. Furthermore, the fungal isolate from the human case with no history of going abroad or immigrating was identified as H. capsulatum var. farciminosum by a gene sequence. These facts indicated that pseudofarcy is not only an infectious disease in horses, but also a zoonotic fungal infection. Japanese autochthonous canine histoplasmosis might be a heteroecism of pseudofarcy because of its likeness to the human case, the similarity of clinical manifestations and the historical background at this stage.

An epidemiological survey of Trichophyton mentagrophytes var. erinacei in the household hedgehog and other rodents was made between January 17, 2002 and February 28, 2002 in Japan. Quills and hairs were collected from sources identified via the internet. The fungus was isolated only from the quills of four-toed hedgehogs (7/18; 39%) from Kanto to Kyushu regions. Isolates were examined morphologically, physiologically and genetically, and identified as T. mentagrophytes var. erinacei anamorph. The isolates were also genetically compared with European hedgehog (Erinaceus europeus)-borne T. mentagrophytes var. erinacei and Kenyan hedgehog (Aterelix albiventris)-borne Arthroderma benhamiae, and their genotypes of the ITS1-5.8S-ITS2 rDNA were all identical. The isolates were crossed with A. benhamiae Americano-European race and African race, A. vanbreuseghemii and A. simii, with the result that they mated only with African race (+) or (-). Mating types of the isolates were (+) in 6 isolates and (-) in one. An intra-isolate mating between one of the 6 plus isolates and the minus one formed abundant mature gymnothesia, the mating type ratio of the F1 progeny was approximately 1:1, and the sib crossings of F1 progeny produced abundant fertile gymnothesia. The present study revealed that the intra-Japanese hedgehog-borne isolate crossing showed complete fertility and that the sexual degeneration pointed out by Takashio (Mycologia 71: 968-976, 1979) did not exist. Two pairs of mating, (+) and (-) mating types of Japanese isolates with (-) and (+) tester strains of A. benhamiae African race formed less gymnothesia, mating type ratios were unbalanced, and sib crossings of F1 progeny produced small gymnothesia containing a low number of asci, pseudogymothesia, or none, respectively. These results show that A. benhamiae var. erinacei, the teleomorph of T. mentagrophytes var. erinacei, belongs to a different mating group (e.g. hedgehog race) than the Americano-European and African races in A. benhamiae.

Thirty-five of 102 extracts obtained from freshly isolated fungal strains showed the antifungal activity against either pathogenic filamentous fungi or pathogenic yeasts. The extract of Aspergillus sp. IFM51759 (Aspergillus versicolor group), which showed the characteristic antifungal activity against Aspergillus fumigatus, one of the major opportunistic human pathogen causing APBA etc., prompted us to isolate antifungal compounds. A new furanone derivative, epimusacin D (1), was isolated along with three known pyranone derivatives, asperlin (2), acetylphomalactone (3), and the diastereomer of asperlin (4). The structure of epimusacin D (1) was determined from the analysis of the spectroscopic investigation. The antifungal activity of the above four compounds were discussed.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. We examined F1 progeny from a crossing between the urease-negative strain (environmental isolate, serotype A, mating type alfa, haploid) and a tester strain (B 3502 from NIH of USA; urease-positive, serotype D, mating type a, haploid) for serotype, mating type, ploidy and urease activity, and performed partial sequencing of the urease gene. Phenotypes of the F1 progeny and results of SSCP analyses suggested that the serotype AD strain of the F1 progeny is a hybrid of the parental serotype A and D strains.

The fungicidal activities of thiocarbamate antifungal agent liranaftate were studied by determining the MIC and the MCC against Trichophyton rubrum with the Milliflex -100 Test System and by determining the time-kill curve in comparison to that of six reference agents. Liranaftate and lanoconazole both showed excellent fungistatic activity against the conidia of T. rubrum. For each of these agents, the MIC after 14 days of contact was 0.009 g/ml. The liranaftate-induced decrease in the MCC occurred from 9 days onwards; MCC at 14 days was 0.039 g/ml. The MCC for tolciclate was also reduced from 9 days onwards, but that of amorolfine, lanoconazole, neticonazole, clotrimazole and bifonazole was not lowered up to 14 days. Similar results were obtained when the studies were performed with germinated conidia. The time-kill curves showed that both liranaftate and tolciclate, at concentrations ranging from 2 to 32 times the MICs, achieved a decrease in viable counts to below the detection limit within 7 to 9 days. In experiments with low levels of inoculum, only amorolfine produced a decrease to below the detection level, and that occurred at 14 days; no reduction in viable counts was observed up to 14 days with the four azole agents. Our data suggest that antifungal agents of the thiocarbamate class possess the most potent fungicidal activity against dermatophytes; these are followed in order by morpholine and azole antifungals.

Aspergillosis has become the biggest cause of death among mycoses in Japan. The main causative agent is Aspergillus fumigatus and its virulence factors have been variously investigated, yet much remains to be clarified. We recently found that the culture filtrate of A. fumigatus has potent anti-leukocyte activity. It seriously damages macrophages and hampers the function of polymorphonuclear neutrophils. The filtrate is also active in vivo: when injected intraperitoneally, the culture filtrate lowered the survival of mice infected with A. fumigatus. This activity becomes overt within 24 hours of culture of the fungus. Although the mechanism of pathogenicity of A. fumigatus is believed to be somewhat complex, we suggest the activities shown in the culture filtrate may play a crucial role in the development of the infection.

Aspergillus fumigatus causes serious, life-threatening human infection, and is one of the most important pathogenic fungi. Little is known, however, about its mechanism of infection or its virulence factors. To learn about its virulence factors, the effect of the culture filtrate of A. fumigatus on macrophages was studied. When cocultured with A. fumigatus in 96-well microplates, murine peritoneal macrophages showed significant morphological changes indicating serious cellular damage, even when the macrophages were not in direct contact with the fungus. Then culture filtrates of Aspergillus spp., A. fumigatus, A. flavus, A. terreus and A. niger, were prepared by culturing the fungus in 96-well or 24-well microplates for 24 h, and the effect of the culture filtrates was determined by culturing macrophages with or without culture filtrate. When cultured with the culture filtrate of A. fumigatus at a concentration of 1% or higher, macrophages demonstrated significant morphological changes, leading to their death. Treatment with heat greatly lowered the activity of the culture filtrate. In contrast, culture filtrates of A. terreus and A. flavus showed no detectable effect on macrophages, whereas A. niger did display a similar, but much weaker effect. Our study strongly suggests that A. fumigatus releases a toxic product (s) in the medium very rapidly, and this may be critically involved as the virulence factor in human infection, at least in part, by causing serious injury to macrophages.

We analyzed a 402-bp sequence of the mitochondrial cytochromebgene of 34 strains ofExophiala jeanselmeiand 16 strains representing 12 related species. The strains ofE.jeanselmeiwere classified into 20 DNA types and 17 amino acid types. The differences between these strains were found in 1 to 60 nucleotides and 1 to 17 amino acids. On the basis of the identities and similarities of nucleotide and amino acid sequences, some strains were reidentified: i.e., two strains ofE.jeanselmeivar.hetermorphaand one strain ofE.castellaniiasE.dermatitidis(including the type strain), three strains ofE.jeanselmeiasE.jeanselmeivar.lecanii-corni(including the type strain), three strains ofE.jeanselmeiasE.bergeri(including the type strain), seven strains ofE.jeanselmeiasE.pisciphila(including the type strain), seven strains ofE.jeanselmeiasE.jeanselmeivar.jeanselmei(including the type strain), one strain ofE.jeanselmeiasFonsecaea pedrosoi(including the type strain), and one strain ofE.jeanselmeiasE.spinifera(including the type strain). SomeE.jeanselmeistrains showed distinct nucleotide and amino acid sequences. The amino-acid-based UPGMA (unweighted pair group method with the arithmetic mean) tree exhibited nearly the same topology as those of the DNA-based trees obtained by neighbor joining, maximum parsimony, and maximum likelihood methods.

Including type strains, mitochondrial cytochrome b genes of 32 strains of Candida albicans and 6 strains of Candida stellatoidea , presently treated as a synonym for C. albicans , were partially sequenced. Analysis of 396-bp nucleotide sequences of the strains under investigation divided C. albicans isolates into three types: type I, type II, and type III; however, strains of C. stellatoidea represented distinct type IV isolates. Deduced amino acid sequences of type I, type II, and type III were identical and differed from that of type IV by one amino acid. Genotypes (rDNA type) of the test strains were also checked. Cytochrome b typing did not correlate with genotyping, and different genotypes occurred for one cytochrome b type. This study shows that cytochrome b gene sequences are useful for analyzing the genetic relatedness of C. albicans isolates and effective for differentiating C. stellatoidea from C. albicans .

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan. To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study. We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P. brasiliensis genes from paraffin-embedded tissue specimens. Successfully amplified were: a 473 base pairs fragment of gp43 gene of P. brasiliensis (located from 741st to 1, 213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P. brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P. brasiliensis yeast cells. The authenticity of the PCR products was confirmed by nucleotide sequence analysis. These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.