Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.     

Keratometry in normal cats: a cross-sectional study in Japan using an automated handheld keratometer

Keratometry was performed in 73 domestic cats of varied signalment in Japan using an automated handheld keratometer. The mean corneal curvature radius was significantly lower for cats younger than 1 year than for those older than 2 years (8.04 mm vs. 8.80−8.99 mm, P<0.01). The radius was significantly greater in males than in females among the cats older than 11 years (9.22 mm vs. 8.84 mm, P=0.01), while the age distributions of the males and females were similar. Corneal astigmatism did not significantly differ across the gender and age groups. The predictability of the corneal curvature and astigmatism was approximately 41−43% and less than 3%, respectively, as a function of age and bodyweight. The results highlighted some age- and sex-related keratometric variations in domestic cats in Japan.     

A case of mycobacteriosis associated with Mycobacterium pseudoshottsii in aquarium-reared fish in Japan

In 2019, several aquarium-reared fish died at a sea life park in Japan. Necropsy revealed micronodules on the spleen in the dotted gizzard shad (Konosirus punctatus). Seven of 16 fish exhibited microscopic multifocal granulomas associated with acid-fast bacilli in the spleen, kidney, liver, alimentary tract, mesentery, gills, and/or heart. Bacterial cultures yielded isolates from the dotted gizzard shad and a Japanese sardine (Sardinops melanostictus). Microbiological and molecular biological examinations revealed the isolates as Mycobacterium pseudoshottsii. To our knowledge, this is the first isolation of M. pseudoshottsii from aquarium-reared fish.     

Effects of intracerebroventricular infusions of corticotropin‐releasing hormone in sheep

To provide new insights into the neural mechanisms of physiological and behavioral responses to stressors in sheep, acute changes in endocrine, autonomic and behavioral functions following 30 min infusions of ovine‐corticotropin‐releasing hormone (oCRH; 0, 0.5, 5 or 50 µg/0.5 mL of artificial cerebrospinal fluid/30 min) into the third ventricle of sheep (n = 7–8) were examined. Serial blood samples were collected through indwelling jugular catheters to determine plasma cortisol concentrations (CORT). Heart rate (HR) and rectal temperature (RT) were obtained via telemetry systems. The behaviors of the animal were monitored simultaneously. Intracerebroventricular infusions of CRH dose‐dependently induced an increase in CORT; there was a time–treatment interaction in CORT (P < 0.001). There was not a time–treatment interaction either in HR (P = 0.29) or in RT (P = 0.28). That RT showed a tendency to decrease with higher doses of CRH in sheep was in contradiction to previous reports in rats and pigs. As to changes in behavioral function, only the induction of bleating was marked. These results suggest that in physiological and behavioral responses of sheep to stressors, CRH regulates the increase in CORT and the induction of bleating. However, CRH might have little function in sympathetic nervous activation during physiological responses to stressors in sheep.     

Reducing effect of dietary anacardic acid on body fat pads in rats

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Toyomizu et al. 2000). In the present study, in order to clarify whether or not anacardic acid could be used advantageously as a special feed/food supplement to reduce fat deposition through the uncoupling action, two sets of experiments were conducted to determine quantitatively the effect of dietary anacardic acid (0.1% w/w) supplementation. More specifically, effects on growth, feed efficiency, fattening, and levels of several constituents of blood serum in rats fed normal and low protein–high carbohydrate (CHO) diets were examined. There were no significant differences in bodyweight gain, feed consumption and feed efficiency among the experimental groups. For the total fat pad content, including inguinal and epididymal fat, significant interaction was shown between both treatments: dietary anacardic acid at 0.1% w/w significantly decreased the total fat pad content in rats fed the CHO diet, but not in rats fed the normal diet. Weights of heart, spleen and brown adipose tissue were not affected by either the dietary treatment or anacardic acid, while both liver and kidney weights decreased with feeding of anacardic acid at 0.1% w/w, but were not affected by the CHO diet. Anacardic acid supplementation in the diet had no effect on serum glutamic oxaloacetic transaminase, alkaline phosphatase or lactate dehydrogenase levels, suggesting that the dysfunction of liver or kidney may not be induced by dietary anacardic acid. The results of the present study reveal a unique function of anacardic acid in that, for dietary conditions enhancing body fat deposition, that is consumption of a diet high in carbohydrates, dietary anacardic acid has the potential to decrease body fat deposition. A possible mechanism for differences observed in anacardic acid‐induced regulation of body fat pad content between rats fed the normal and CHO diets, based on uncoupling action of anacardic acid on the mitochondrial oxidative phosphorylation, is discussed.     

Seasonal changes of the mineral contents in the rumen of wild Yeso sika deer (Cervus nippon yesoensis)

The rumen contents were collected from 36 wild Yeso sika deer (Cervus nippon yesoensis) captured by deer culling or by hunting in the spring, summer, autumn and winter in Hokkaido, Japan. Botanical classification was conducted, and the contents of mineral (calcium (Ca), phosphorus (P), potassium (K), sodium (Na), iron (Fe), copper (Cu) and zinc (Zn)) were measured. The animals were captured around pastures or fallow field areas in the Kushiro area. The rumen contents consisted of grasses and Sasa sp. leaves regardless of the season. Leaves and bark were ingested in the spring, autumn and winter. The macro‐mineral contents in the rumen showed seasonal changes. In the summer, the Ca, K and P contents were high, and the Na content was low. There were no seasonal changes in the Fe content. The P, Na and Fe contents were higher than the animals' requirements. In a future survey, it is needed to determine the mineral contents of the food ingested by wild Yeso sika deer.     

Linkage mapping of four chicken calpain genes

Calpains are intracellular Ca²⁺‐dependent proteases and enzymes that contribute to growth and meat quality. In the present study, we identified polymorphisms in four calpain genes (CAPN1, CAPN2, CAPN3, and CAPN1.5) expressed ubiquitously in chicken using polymerase chain reaction‐restriction fragment length polymorphism, and mapped them using two backcross families (East Lansing (EL) and Kobe University (KU)). CAPN2 and CAPN1.5 mapped to two locations on chromosome 3 about 30 cM apart, while CAPN3 mapped to chromosome 5. CAPN1 was linked to a previously unlinked microsatellite marker LEI0140 to form a new linkage group called E66. CAPN2 and CAPN3 extend the amount of conserved synteny between chicken chromosome 3 and human chromosome 1, and between chicken chromosome 5 and human chromosome 15, respectively. Although CAPN2, CAPN3, and CAPN1.5 were found in the University of California Santa Cruz chicken genome browser gateway, CAPN1 and LEI0140 were not in specific genomic positions.     

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.