High-grade myofibroblastic sarcoma of inguinal region in a dog

A subcutaneous tumor in the left inguinal region was present in an 11-year-old female bloodhound. Histopathologically, the tumor showed invasive growth and extensive necroses, and it was composed of spindle-shaped, elongated, and stellate neoplastic cells accompanied by occasional giant cells arranged in fascicular, herringbone, or irregular storiform patterns with abundant production of collagen fibers. The cytoplasm of most tumor cells was positive for vimentin, alpha-smooth muscle actin, and calponin, but was negative for desmin, smoothelin, and S-100. Furthermore, most of the tumor cells were negative for Iba1 while some tumor cells were weakly positive. Thus, this tumor was diagnosed as a high-grade myofibroblastic sarcoma according to the diagnostic criteria for human myofibroblastic sarcomas.     

Feline demodicosis caused by an unnamed species

A case of feline demodicosis is described in this report. A 13-year-old spayed female domestic short hair cat weighing 4.5 kg was being treated with cefovecin and alternately with prednisone or methylprednisolone. On further physical examination, the cat showed mild erythema and hair loss on the bridge of the nose, around the eyes, on the chin, on the side part of the breast and on the abdomen. A large number of Demodex mites were found in deep skin scrapings from the affected areas. The cat was then treated with ivermectin at 600 μg/kg administered SC daily. After 4 weeks of treatment, the cat was clinically normal with no mites detected in the skin scrapings from the face or breast areas. The mite responsible may represent a previously seen but as yet unnamed new species. This is third report that describes a case of feline demodicosis caused by a different, unnamed mite species that has different morphological characteristics to those of known Demodex mites and may represent a previously seen but as yet unnamed species.     

High prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 from cattle in selected regions of Japan

The prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 was examined in bovine faeces. EHEC O157 was isolated from the faeces of 42 (13.0%) of 324 cattle. Of the 4 farms and the facilities tested, the 3 farms and the facilities were found positive for EHEC O157. The highest isolation rate among the farms was 33.7%. The prevalence of EHEC O157 in heifers was higher than that in calves and other cattle. No cattle positive for EHEC O157 showed any clinical signs except 2 calves with diarrhea in a veterinary hospital. Almost all isolates possessed the stx gene, and Stx-positive strains carrying both stx(1) and stx(2) genes were predominant. These results indicate that EHEC O157 are distributed in bovine faeces, and that dairy and beef farms in selected regions of Japan are heavily contaminated with the organisms.     

Trial for detecting carriers with major genes in a selected layer line

Production traits of a Rhode Island Red layer line were analyzed to detect carrier animals with major genes that influenced a particular trait by applying the major gene index (MGI) approach. This method was used to examine the deviation of predicted breeding values between parents and offspring. Advantages of this method include its simplicity and time‐reducing, cost‐saving benefits compared with any other statistical approach for screening major genes. The layer line had been selected based on the desired gain index for 6 years at the National Livestock Breeding Center, Okazaki station. The line consisted of 125 sires and 2986 dams. Two economic traits as breeding objectives were studied – age at sexual maturity (ASM, days) and egg production efficiency (EP, %). The MGI detected nine sires and 23 dams as carriers with major genes for ASM, and five sires and 26 dams for EP. They were identified as important breeding stock, and pedigree information provided candidates possessing major genes with favorable effect at the founding of the selected layer line. It seems likely that the simple approach of the MGI could be a useful preliminary method for detecting carrier animals with major genes before applying molecular techniques on a sampled population to identify in more detail the existence of major genes and their carriers.     

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

Establishing conditions for the storage and elution of rabies virus RNA using FTA? cards

The Flinders Technology Associates filter paper cards (FTA^® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA^® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA^® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA^® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA^® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA^® cards.     

Pacific Ocean and Japan Sea ecotypes of Japanese beech (Fagus crenata) differ in photosystem responses to continuous high light

Two ecotypes of Japanese beech (Fagus crenata Blume), the Pacific Ocean type (PAO) and the Japan Sea type (JAS), show different responses to high solar irradiance. When PAO and JAS saplings were grown in continuous high-light (H), leaves of JAS became pale green. To elucidate this phenomenon, we investigated in vivo photochemistry based on pigment concentrations of Photosystem (PS) I and PS II and Western blot analysis. In JAS-H leaves, the amount of D1-protein decreased, resulting in decreases in the maximal quantum yield of PS II (F(v)/F(m)) and electron transport rate, whereas PAO-H leaves maintained high activities. The PS I photochemistry determined by measurement of P-700 photo-oxidation showed that the intersystem electron pool size was 1.4 times greater in JAS-H leaves than in PAO-H leaves. Furthermore, the re-reduction kinetics of P-700(+) showed that cyclic electron transport around PS I was 1.2 times faster in PAO-H leaves than in JAS-H leaves. Analysis of the area over the fluorescence induction kinetics indicated that the relative abundance of the PS IIalpha center increased in PAO-H leaves, whereas JAS leaves were observed to have low acclimation capacity to high light. These results demonstrate that PAO leaves possess acclimation mechanisms to continuous high light, whereas JAS leaves are more vulnerable to continuous high light, resulting in reduced leaf longevity owing to photoinhibition caused by increases in the intersystem electron pool size and suppression of photochemistry at the level of PS I and PS II.     

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F₂ progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.