No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8)

Background

Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission.

Methods

Three pairs of animals consisting of a dog inoculated with CIV (10^8.3 egg infectious dose₅₀/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia.

Results

All three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses.

Conclusions

These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.     

Isolation and genetic analysis of Japanese encephalitis virus from a diseased horse in Japan

Japanese encephalitis (JE) developed in an unvaccinated half-bred horse kept in Tottori Prefecture, Japan. The animal showed ataxia with pyrexia and low appetite, and ultimately died. A viral strain was isolated from the cerebrum of the horse and was identified as JE virus (JEV) by RT-PCR using JEV specific primers. The isolated JEV was classified into genotype I by nucleotide sequence analysis of the viral envelope gene. We believe that this is the first report of the genotype I strain being isolated from a horse.     

Efficacy of oseltamivir phosphate to horses inoculated with equine influenza A virus

We investigated the efficacy of the oral administration of oseltamivir phosphate (OP) in horses experimentally infected with equine influenza A virus (H3N8). Nine horses were divided into three horses each of control, treatment and prophylaxis groups. An administration protocol for the treatment group (2 mg/kg of body weight, twice a day for five days) was started immediately after the onset of pyrexia (above 38.9 degrees C). An administration protocol for the prophylaxis group (2 mg/kg of body weight, once a day for five days) was started on a day before viral inoculation. In the treatment group, periods of virus excretion (mean days +/- standard deviation, 2.3 +/- 0.6) and pyrexia (2.0 +/- 0.0) were apparently shorter than those of the control group (6.0 +/- 0.0 and 8.0 +/- 1.0, respectively). In the prophylaxis group, although virus excretion and pyrexia were not prevented, the periods of virus excretion (5.0 +/- 0.0) and pyrexia (4.7 +/- 1.5) were shorter than those of the control group. Moreover, in the treatment and prophylaxis groups, bacterial counts of Streptococcus equi subsp. zooepidemicus known as the common pathogen of secondary bacterial pneumonia in bronchoalveolar lavage fluids collected seven days after inoculation were significantly fewer than that of the control group. The results indicated that the oral administration of OP to horses affected with equine influenza would contribute to reduce the magnitude of virus excretion, pyrexia and consequent secondary bacterial pneumonia.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

Plasma concentrations of immunoreactive (ir)-inhibin, gonadotropins and steroid hormones during the ovulatory cycle of the duck

The present study was undertaken to determine changes in circulating levels of immunoreactive (ir)-inhibin, FSH, LH, estradiol-17beta, progesterone, and testosterone during the ovulatory cycle of Shao ducks. Serial blood samples were taken from two groups of laying ducks for measurement of ir-inhibin, gonadotropins, and steroid hormones at 2 h intervals for 24 h. Plasma concentrations of ir-inhibin did not change significantly during the ovulatory cycle. The highest level of plasma ir-inhibin was observed 6 h prior to ovulation, which coincided with a decreased level of plasma FSH. One FSH surge was found 12 h after ovulation. Estradiol-17beta, progesterone, and testosterone were also determined during the ovulatory cycle. Two peak values were detected for estradiol-17beta 8 h before ovulation and 4 h after ovulation, while progesterone started to increase 4 h before ovulation and reached a peak at ovulation. The highest level of plasma testosterone was detected around the time of ovulation. These results suggest that inhibin may be involved in the control of FSH secretion during the ovulatory cycle. In addition, both LH and progesterone are of importance in the ovulation process of Shao ducks.     

Immunolocalization of inhibin/activin subunits in the shiba goat fetal, neonatal, and adult testes

The objective of this study was to investigate the cellular immunolocalization of inhibin alpha and inhibin/activin (betaA and betaB) subunits in the fetal, neonatal and adult testes of Shiba goats. The testes were obtained from a fetus at 90 days, a neonate at 15 days, and two adult Shiba goats (both of 3 years old). The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA, and inhibin/activin betaB. Inhibin alpha and inhibin/activin (betaA and betaB) subunits were expressed in Leydig cells, but not in the Sertoli cells of the fetus with a weak immunostaining. An increase in the number of positive cells and a more intense immunohistochemical signal for inhibin alpha and inhibin/activin (betaA and betaB) subunits were observed in the Leydig cells of neonatal testes. Moreover, inhibin alpha, betaA, and betaB subunits were expressed in the Sertoli cells and Leydig cells of adult testes, respectively. These results suggest that Shiba goats testes have the ability to synthesize inhibins in the fetus, neonate, and adult, and the cellular localization of inhibin/activin subunits showed age-related changes in fetal, neonatal, and adult testes of Shiba goats.     

Annual changes in testicular size and serum and fecal testosterone concentrations in male bharals, Pseudois nayaur

The serum and fecal testosterone (T) concentrations and testicular sizes of two male bharals (Pseudois nayaur) were determined for approximately one year. The profiles of the fecal T concentrations showed a similar tendency as the profiles of serum T concentrations, and there was a significant correlation between serum and fecal T concentrations (r=0.72). T concentrations rose drastically in October and decreased gradually until January. The maximum testicular size was observed between November and January. Semen collected between December and January was excellent in quality and comparable to domestic sheep and goats. The active periods of the testes were synchronized with the early breeding season of females.     

Grade of weathering and fertility of volcanic ash soils of Aso volcano

Texture is one of the major criterions in soil classification, probably because it has a decisive influence on soil properties. This is particularly true for volcanic ash soils. Most ashes are largely composed of sand and silt particles with little clay (11, 15). The ash weathers very rapidly (1), and clay site particles less than 2 microns in diameter occur even within a few months, as shown by Ishii at the authors' laboratory. Those clay size particles produced in the early stage of weathering are slightly weathered ones (2), and are still subject to rapid weathering, losing bases and silica under humid and well drained conditions. In consequence the clay fraction of volcanic ash soils is composed of particles which vary in degree of weathering from slightly altered glass and feldspar to true clay mineraloids and minerals. The clay fraction of younger soils as a whole is less and that of older ones is more weathered. Weathering brings a remarkable change in the properties of volcanic ash soils; for example, an inerease in soil acidity, lowering of base saturation and bulk density, or accumlation of organic matter. These changes must exert a great influence on soil fertility directly or indirectly.     

The Vigna angularis complex: Genetic variation and relationships revealed by RAPD analysis, and their implications for in situ conservation and domestication

The present study, using RAPD analysis, was undertaken to characterize genetic variation in three forms of V. angularis, cultivated, wild and weedy forms, and their relationships. The materials used consisted of 171 individuals (plants) or cultivars from 23 populations including 5 wild populations, 6 weedy populations, 6 cultivated populations and 6 populations with plants having wild and weedy or intermediate morphology, denoted here as complex populations. The materials used were collected on Honshu Island, Japan and seeds collected directly from the field were germinated for DNA extraction. In addition, 6 landrace accessions of V. angularis from the genebank were also analyzed. Genetic variation was highest in the wild form (H_g= 0.132; GD = 0.388), followed by the weedy form (H_g= 0.124; GD = 0.341) and the least in the cultivated form (H_g= 0.079; GD = 0.274). Intra-population genetic variation was high in the weedy and in the wild populations. However, inter-population was greater than intra-population genetic variation for all groups of populations studied in the V. angularis complex. 93% of the total diversity in the present study was exhibited by plants from complex populations and specific RAPD bands were found in these populations. Our results provide evidence that complex populations would be a logical focus for efforts to conserve the V. angularis complex in situ. Our results suggest that weedy populations are usually an ecotype of the wild form adapted to a different habitat.     

Simultaneous determination of neonicotinoid insecticides in agricultural samples by solid-phase extraction cleanup and liquid chromatography equipped with diode-array detection

Effective sample pretreatment procedures based on solid-phase extraction (SPE) for multiresidue determination of seven neonicotinoid insecticides in agricultural products were investigated. After extraction with acetone and concentration, the insecticides in aqueous sample extracts were transferred into organic solvent phases with a Chem Elut SPE cartridge. Finally, the eluate from the cartridge was cleaned up with a SPE cartridge packed with graphitized carbon black and aminopropyl silica gel, which showed a higher cleanup efficiency than the classical silica gel SPE cartridge. Seven insecticides were separated on a reversed-phase C18 column and a gradient system of methanol and phosphate solution based on high-performance liquid chromatography. The established multiresidue determination has been applied to several artificially spiked agricultural samples, with the result that the average recoveries were excellent, with the exception of nitenpyram. The limit of detection of the method ranged from 0.01 to 0.03 mg/kg for the insecticides.