<Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype (<Emphasis Type="Italic">A. mali</Emphasis>) causes black spot of European pear

A disease caused by Alternaria alternata occurred on the leaves of European pear cultivar Le Lectier in Niigata Prefecture, Japan, and was named black spot of European pear. In conidial inoculation tests, the causal pathogen induced not only small black lesions on the leaves of European pear cultivar Le Lectier, but severe lesions on the leaves of apple cultivar Red Gold, which is susceptible to the A. alternata apple pathotype (previously called A. mali) causing Alternaria blotch of apple. Interestingly, the apple pathotype isolate showed the same pathogenicity as the European pear pathogen. HPLC analysis of the culture filtrates revealed that A. alternata causing black spot of European pear produced AM-toxin I, known as a host-specific toxin of the A. alternata apple pathotype. AM-toxin I induced veinal necrosis on leaves of Le Lectier and General Leclerc cultivars, both susceptible to the European pear pathogen, at 5?×?10^?7 M and 10^?6 M respectively, but did not affect leaves of resistant cultivars at 10^?4 M. PCR analysis with primers that specifically amplify the AM-toxin synthetase gene detected the product of expected size in the pathogen. These results indicate that A. alternata causing black spot of European pear is identical to that causing Alternaria blotch of apple. This is the first report of European pear disease caused by the A. alternata apple pathotype. This study provides a multiplex PCR protocol, which could serve as a useful tool, for the epidemiological survey of these two diseases in European pear and apple orchards.     

Neutropenia associated with osteomyelitis due to Hepatozoon canis infection in a dog

A 4-year-old, intact male Shiba dog was referred to Yamaguchi University Animal Medical Center, Yamaguchi, Japan, for the following complaints: anorexia, lethargy, intermittent fever, gingival bleeding and abdominal purpura. The dog presented with persistent neutropenia. Histopathological examination of a bone marrow sample revealed round to oval structures that resembled Hepatozoon micromerozoites and formed a "wheel-spoke" pattern. Furthermore, mature neutrophils were observed around these structures. PCR and sequencing using bone marrow aspirate confirmed Hepatozoon canis (H. canis) infection. These findings suggest that the neutropenia observed in this case was associated with osteomyelitis due to H. canis infection. This is the first report of neutropenia associated with H. canis infection. H. canis infection can be included in the differential diagnosis in canine cases of neutropenia in areas where the disease is endemic.     

Direct evidence of multiple paternities in natural population of viviparous Japanese surfperch by allelic markers of microsatellite DNA loci

This study was performed to obtain information on the occurrence of multiple paternities in three species of viviparous Japanese surfperch using allelic markers of microsatellite DNA loci. Direct evidence for multiple fertilizations was established by reconstructing paternal genotypes from the progeny of gravid females. Multiple paternities were ascertained in five of 10 broods of Ditrema temmincki and in three of nine broods of Neoditrema ransonneti, but not in Ditrema viride. The number of patrilines detected in the progeny of D. temmincki and N. ransonneti females were two or three, respectively, as determined by the GERUD v2.0 algorithm for reconstructing parental genotypes from half-sib progeny arrays.     

Cytotoxic constituents in the holdfast of cultivated Laminaria japonica

ABSTRACT:     Four steroidal ketones were isolated from the holdfast of cultivated Laminaria japonica . The structures were characterized as ergosta-4,24(28)-diene-3-one (1), ergosta-4,24(28)-diene-3,6-dione (2), stigmasta-4,24(28)-diene-3-one (3) and stigmasta-4,24(28)-diene-3,6-dione (4), by spectral data. Compounds 2 and 4 were shown to be cytotoxic against the MCF-7 human breast cancer cell, and the growth of MCF-7 was inhibited by 96% and 79%, respectively, at 10 µg/mL. It is the first report on the isolation of cytotoxic steroidal ketones from the kelp in the genus of Laminaria.     

Characterization of Bovine herpesvirus type 4 isolated from cattle with mastitis and subclinical infection by the virus among cattle

An outbreak of contagious mastitis occurred among cattle on a farm, and bovine herpesviruses were isolated from the affected mammary tissues, scabs and abscess discharge of the cattle. A bovine herpesvirus type 4 (BoHV-4)-specific fragment was amplified from the isolates by polymerase chain reaction (PCR). Restriction endonuclease analyses demonstrated that the isolates were related to Movar-like European type BoHV-4. To determine the ratio of BoHV-4 subclinical infection in the cattle, a genomic survey was performed by PCR for cattle that were moved to the animal hygiene service station in Ibaraki prefecture. The BoHV-4 genome was occasionally detected in peripheral blood leukocytes, lymph nodes and nervous tissues. The rate of BoHV-4 subclinical infection was relatively high in the cattle.     

Incidence of canine glaucoma with goniodysplasia in Japan : a retrospective study

The incidence of primary and secondary glaucoma in dogs was investigated. A total of 1244 dogs received ophthalmologic examinations, including tonometry and gonioscopy. Goniophotographs were taken using a goniolens to evaluate the iridocorneal angle (ICA) as well as pectinate ligament (PL). The anterior width of the ciliary cleft and the total distance from the origin of the PL to the anterior corneal surface were measured from the goniophotographs. Glaucoma was diagnosed based on the cupping of the optic nerve head, clinical signs, ocular changes, and high IOP, and it was synchronized with gonioscopic grades to differentiate between primary and secondary glaucoma. We investigated 1244 dogs of 29 breeds, including the mixed breed; among these, glaucoma was diagnosed in 127 dogs (162 eyes). Of 162 eyes, primary glaucoma was diagnosed in 129 eyes and secondary glaucoma in 33 eyes. Shiba Inu dogs (42 dogs, 33%) showed the highest incidence of glaucoma, followed by Shih-Tzu (21 dogs, 16.5%). Furthermore, all the glaucomatous Shiba Inu dogs had primary glaucoma with abnormal ICA grades and dysplastic PLs. The findings of our study reveal that the Shiba Inu breed in Japan may have a hereditary predisposition to glaucoma.     

Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.     

Detection of carrot red leaf virus-RNA in carrot seeds by multiplex RT-nested PCR

A practical method to detect carrot red leaf virus (CtRLV)-RNA in carrot seeds using a multiplex RT-nested PCR was developed. Total RNA was extracted from carrot seeds by the guanidine thiocyanate method. RT-PCR was performed with a one step RT-PCR kit with CtRLV primers and carrot ubiquitin primers for the internal control, and the second PCR was performed with CtRLV nested primers. Ubiquitin were detected by the first PCR in all samples, while distinct CtRLV-RNA was detected by the 2nd PCR. CtRLV-RNA was detected from carrot seeds from three cultivars. The multiplex RT-nested PCR method was able to detect CtRLV-RNA from a single carrot seed.     

Choroidal melanoma in a dog

A 7-year-old intact female golden Retriever was referred for evaluation of an intraorbital mass of the left eye. Based on ophthalmoscopy, ultrasonography and magnetic resonance imaging (MRI), the tentative diagnosis was made as an intraocular neoplasia, especially choroidal melanoma. The orbital exenteration of the affected eye was performed. The mass was histologically diagnosed as malignant choroidal melanoma. No signs of recurrence and metastasis were detected by thoracic radiographs, blood examinations and MR images, and the dog was clinically healthy for 23 months after operation.     

A canine pemphigus foliaceus case showing parallel relationship of disease activity and titer of serum anti-keratinocyte cell surface antibodies

A seven-year-old, spayed female mongrel dog was diagnosed as pemphigus foliaceus (PF) by clinical, histopathological and immunopathological observations. Serum antibodies against the cell surface of keratinocytes in the dog were detected by indirect immunofluorescence (IIF) using cryosectioned bovine esophagus as well as living cultured-canine keratinocytes as the substrates. When we compared the titers of IIF on bovine esophagus with its disease activity, the IIF titers reflected the disease activity throughout the time course. Our findings will suggest that sequential titration of serum antibodies by IIF will be useful for monitoring the serological disease activity in canine PF.