Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.     

Relationship between hybridization frequency of Brassica juncea × B. napus and distance from pollen source (B. napus) to recipient (B. juncea) under field conditions in Japan

Several imported transgenic canola (Brassica napus) seeds have been spilled and have grown along roadsides around import ports. B. juncea, a relative of B. napus with which it has high interspecific crossability, is widely distributed throughout Japan. There is public concern about the harmful impacts of feral B. napus plants on biodiversity, but spontaneous hybridization between spilled B. napus and weedy B. juncea populations is hardly revealed. We evaluated the relationship between the hybridization frequency of B. juncea × B. napus and their planting distance in field experiments using the mutagenic herbicide-tolerant B. napus cv. Bn0861 as a pollen source for hybrid screening. The recipient B. juncea cv. Kikarashina was planted in an experimental field with Bn0861 planted in the center. No hybrids were detected under natural flowering conditions in 2009. However, the flowering period was artificially kept overlapping in 2010, leading to a hybridization frequency of 1.62% in the mixed planting area. The hybridization frequency decreased drastically with distance from the pollen source, and was lower under field conditions than estimated from the high crossability, implying that spontaneous hybridization between spilled B. napus and weedy B. juncea is unlikely in the natural environment.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Deep granulomatous dermatitis of the fin caused by Fusarium solani in a false killer whale (Pseudorca crassidens)

A 10-year-old female false killer whale (Pseudorca crassidens) developed skin lesions in the left breast fin. Histopathologically, the lesions consisted of multiple granulomas spread diffusely into the deep dermis and bone; characteristically, each granuloma had septate, branching fungal hyphae and chlamydospores surrounded by eosinophilic Splendore-Hoeppli materials. Macrophages, epithelioid cells and multinucleated giant cells in the granulomas reacted mainly to anti-SRA-E5 antibody against human macrophage scavenger receptor type I. Fusarium solani was isolated and its gene was detected from the skin samples. Mycotic skin lesions by Fusarium spp. reported so far in marine mammals were regarded as superficial dermatitis; therefore, the present case is very uncommon in that the lesions spread deeper into the skin.     

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.     

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.