Role of actin microfilaments in canine distemper virus replication in vero cells

Several studies have indicated that viruses require a specific cytoskeletal structure for replication in host cells. In this study, we examined the role of actin fiber in the replication of canine distemper virus (CDV), belonging to the Morbillivirus genus of the family Paramyxoviridae. For this purpose, we used two actin depolymerizing agents, cytochalasin-D (C-D) and mycalolide-B (ML-B). In Vero cells, C-D disrupted actin fibers distributed in the cytosol, but peripheral actin fibers remained intact. On the other hand, ML-B completely disrupted the actin fibers distributed in both areas. Treatment of Vero cells with C-D or ML-B inhibited the replication of CDV. Double staining of CDV-infected Vero cells with antibody to N-protein and rhodamine-phalloidin revealed the presence of N-protein in mid-cytoplasm. However, the N-protein was specifically localized at the submembrane region in the presence of C-D, whereas it was clustered in the presence of ML-B. Viral mRNA levels of N- and H-proteins were rather increased by treatment with C-D or ML-B. The treatment with ML-B strongly inhibited N-protein expression, whereas C-D only slightly inhibited N-protein expression. These results suggest that actin microfilaments distributed in the cytoplasm and on the membrane region in host cells may have a different role in the process of CDV replication.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

Identification of Card15/Nod2 mRNA in intestinal tissue of experimentally induced colitis in rats

Card15/Nod2 has been suggested to be an intracellular pathogen-associated molecular pattern (PAMPs) recognition molecule, which contains a leucine-rich repeat region similar to the Toll-like receptors (TLRs). Card15/Nod2 gene variants play an important role in the susceptibility to Crohn's disease. In this study, we examined the kinetics of Card15/Nod2 expression in intestinal tissue during inflammation in the 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-treated rat experimental colitis model. At 2 and 4 days after TNBS administration, the mononuclear cells remarkably infiltrated the mucosal layer and tunica muscularis, which was followed by a gradual decrease to resting levels at 14 days after TNBS administration. Card15/Nod2 mRNA expression increased and peaked at 4 days after the TNBS administration, followed by a gradual decrease in accordance with the amelioration of the inflammatory response. Expressions of Tlr2, Tlr4 and Myd88 were also upregulated in the inflamed colonic region, and in an in situ hybridization study, a positive signal for Card15/Nod2 was observed in the crypt of the epithelial cell layer and in the infiltrated cells of the submucosal and myenteric regions. These results suggest that in addition to the TLR recognition systems, Card15/Nod2 may contribute to the inflammatory process not only in the epithelial and submucosal layers but also in the tunica muscularis.     

Induction of estrus by prostaglandin F2alpha administration in the vaginal vestibules of miniature pigs

Estrus induction is an important step in embryo production. It has been difficult to induce estrus in miniature pigs by intramascular (i.m.) injection of prostaglandin F2alpha (PGF2(2alpha)) in the early luteal stage of the estrous cycle. In the present study, we injected two different doses of PGF2(2alpha) i.m. and into the submucosa of the vaginal vestibule (i.ves.) of miniature pigs, and examined the effect of these treatments on estrus induction. Fifteen miniature pigs were divided into five experimental groups (control, saline injected i.m.; PGF2alpha treated, 1.0 or 1.5 mg of PGF2alpha injected twice i.m. or i.ves.), and the estrus length and concentrations of 17beta-estradiol (E2) and progesterone (P) in the blood were examined. Estrus length was significantly shortened by a large amount of PGF2alpha injected i.ves. In addition, the concentration of P in the blood significantly decreased after two injections of PGF2alpha (i.m. or i.ves.). These results suggest that in miniature pigs, administration of at least 3.0 mg of PGF2alpha is required for the induction of luteolysis and injection of PGF2alpha into the vaginal vestibule is a useful method of estrus induction.     

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat,Fagopyrum homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.     

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

Background

Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.

Findings

After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.

Conclusions

These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.     

Successful induction of lactation in a barren thoroughbred mare: growth of a foal raised on induced lactation and the corresponding maternal hormone profiles

The purpose of this study was to demonstrate that a barren parous Thoroughbred mare with lactation induced by hormonal treatment can be introduced to an orphan foal at the same farm and that the mare can become pregnant after the end of the hormonal treatment. An additional purpose was to investigate the changes in the plasma concentrations of prolactin, estradiol-17β, progesterone, follicle-stimulating hormone, and luteinizing hormone before, during, and after hormonal treatment. The difference in body weight between the adopted foal and the control foals, which were at the same farm and raised by their natural mothers, was 17 kg at 24 weeks old, when the foals were weaned. However, the adopted foal and the control foals had almost the same weight at 35 weeks old and later. The first ovulation after hormonal treatment was confirmed 10 days after the end of hormonal treatment and then the normal estrous cycle resumed. Furthermore, the changes in plasma progesterone, estradiol-17β, follicle-stimulating hormone, and luteinizing hormone showed regular patterns after the first ovulation. Conception was confirmed in the fifth ovulation. Meanwhile, another study demonstrated that conception was confirmed in the first ovulation after hormonal treatment. The present study is the first to demonstrate the hormonal profiles during and after induction of lactation in a Thoroughbred mare. This approach is useful for solving the economic and epidemic problems of introducing a nurse mare to an orphan foal.     

Factors Influencing the Frequency of Pregnancy Loss among Thoroughbred Mares in Hidaka,Japan

Pregnancy loss in mares is thought to be a main problem associated with reproductive efficiency. To clarify the situation of pregnancy loss in Thoroughbred mares in Japan, the occurrence of pregnancy loss before and after 35 days of gestation was investigated with 1,476 Thoroughbred mares in Hidaka, Japan, from 2007 to 2009. Pregnancy loss on days 17-35 was determined by ultrasound examination between 17 and 35 days after the last mating. Follow-up surveys were conducted between 35 days and foaling to determine pregnancy loss on day 35 until foaling in 843 of these mares. Using multiple logistic regression analysis, we assessed the influence of mare age, reproductive status, twin pregnancy reduction, body condition score (BCS), estrus type in foaling mares (foal heat or not), progesterone therapy, and endometrial cysts on pregnancy loss rates on days 17-35 and on day 35 until foaling in this population of mares. The pregnancy loss rates on days 17-35 and on day 35 until foaling were 5.8% and 8.7%, respectively. The overall pregnancy loss rate (day 17 until foaling, including parturient losses) was 14.7%. Risk factors for pregnancy loss included decrease in BCS between 17 and 35 days, <5 BCS at day 35, mating during foal heat, and endometrial cysts. In all, 14.7% of pregnancies were lost between day 17 and birth, contributing significantly to reduced reproductive efficiency in Thoroughbred mares in Japan. These observations indicate that mares should be maintained at high BCS and should be prevented from mating in foal heat to decrease the pregnancy loss rates.     

Empirical Percentile Growth Curves with Z-scores Considering Seasonal Compensatory Growths for Japanese Thoroughbred Horses

Percentile growth curves are often used as a clinical indicator to evaluate variations of children’s growth status. In this study, we propose empirical percentile growth curves using Z-scores adapted for Japanese Thoroughbred horses, with considerations of the seasonal compensatory growth that is a typical characteristic of seasonal breeding animals. We previously developed new growth curve equations for Japanese Thoroughbreds adjusting for compensatory growth. Individual horses and residual effects were included as random effects in the growth curve equation model and their variance components were estimated. Based on the Z-scores of the estimated variance components, empirical percentile growth curves were constructed. A total of 5,594 and 5,680 body weight and age measurements of male and female Thoroughbreds, respectively, and 3,770 withers height and age measurements were used in the analyses. The developed empirical percentile growth curves using Z-scores are computationally feasible and useful for monitoring individual growth parameters of body weight and withers height of young Thoroughbred horses, especially during compensatory growth periods.