A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats

Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT‐EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 m PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.     

Immunohistochemical analysis of cyclooxygenase-2 induction during wound healing in dog skin

Cyclooxygenase (COX)-2 is an inducible isoform of COX and is expressed under abnormal health conditions. This study elucidated the cutaneous induction of COX-2 during the wound healing processes in dog skin. Dog skin was sutured after punch biopsy and investigated histologically and immunohistochemically on days 0 (normal), 1, 3, 5, 7, 10, and 14 after injury. Histological changes, including infiltration of inflammatory cells and proliferation of fibroblast-like cells, were observed as predicted, and there was a close and significant correlation between these 2 events. COX-2-positive cells were detected in the epidermis between days 1 and 7, and bimodal peaks were observed in the case of the percentage of COX-2-positive cells. In inflammatory cells, COX-positive signals were detected on day 3 only. Here, we clarified the localization and pattern of the induced COX-2 expression during wound healing in dog skin.     

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.     

Ectomycorrhizal fungal communities of Dipterocarpus alatus seedlings introduced by soil inocula from a natural forest and a plantation

We attempted to introduce ectomycorrhizal (ECM) fungi onto seedlings of Dipterocarpus alatus (Dipterocarpaceae) by soil inocula collected from tree stands of D. alatus. Top soils collected from beneath the trees of D. alatus in a dry evergreen forest and a 15-year-old plantation were inoculated onto germinating seeds of D. alatus. After 7 months of seedling cultivation, ECM fungal communities in the seedlings were investigated based on the sequences of ITS rDNA. The ECM fungi detected were divided into 19 phylotypes by molecular analysis. Most of the phylotypes were identified as ECM fungal taxa, i.e., Clavulina, Laccaria, Lactarius, Tomentella, Pyronemataceae, and Tricholomataceae. Accordingly, we can confirm that soil inoculation is a simple method to induce ECM formation with diverse fungi in pot cultured seedlings, which would be useful for introducing diverse ECM fungi to dipterocarp plantations.     

Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae

The membrane rotor ring from the vacuolar-type (V-type) sodium ion-pumping adenosine triphosphatase (Na+-ATPase) from Enterococcus hirae consists of 10 NtpK subunits, which are homologs of the 16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F1Fo- or F-ATPases, respectively. Each NtpK subunit has four transmembrane alpha helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.     

Length of lags in responses of milk yield and somatic cell score on test day to heat stress in Holsteins

We used daily records from provincial Japanese weather stations and monthly test‐day records of milk production to investigate the length of the lags in the responses of cows’ milk yield and somatic cell score (SCS) to heat stress (HS). We also investigated the HS thresholds in milk yield and SCS. Data were a total of 17,245,709 test‐day records for milk and SCS in Holstein cows that had calved for the first time between 2000 and 2015, along with weather records from 60 weather stations. Temperature–humidity index (THI) values were estimated by using average daily temperature and average daily relative humidity. Adjusted THI values were calculated by using temperature, relative humidity, wind speed, and solar radiation. The model contained herd, calving year, month of test day, age group, days in milk, and THI as a fixed effect. THIs for each day from 14 days before the test day until the test day were used to represent the HS effects. The HS occurring 3 days, and between 8 and 10 days, before the test day had the greatest effect on the milk yield and SCS, respectively. The threshold THI values for the HS effect were about 60–65 for both traits.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

Retrospective diagnosis of feline GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japan: possible spread of the mutant allele in the Japanese domestic cat population

GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disease caused by simultaneous deficiencies of acid beta-hexosaminidase (Hex) A and Hex B due to an abnormality of beta-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. In the present study, a retrospective diagnosis was performed in 2 previous suspected cases of feline Sandhoff-like disease using a DNA test to detect the causative mutation identified previously in 4 cats in 2 other families of Japanese domestic cats. Enzymic analysis was also performed using stored leukocytes and plasma collected from the subject families in order to investigate the usefulness of enzymic diagnosis and genotyping of carriers. The DNA test suggested that the 2 cases were homozygous recessive for the mutation. Consequently, 6 cats homozygous for the same mutation have been found in 4 separate locations of Japan, suggesting that this mutant allele may be spread widely in the Japanese domestic cat populations. In enzymic analysis, Hex A and Hex B activities in leukocytes and plasma measured using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate were negligible in affected cats, compared with those in normal and carrier cats. However, there was a wide overlap in enzyme activity between normal and carrier cats. Therefore, it was concluded that enzymic analysis is useful for diagnosis of affected cats, but is not acceptable for genotyping of carriers.     

Bacillary hemoglobinuria in Japanese black cattle in Hiroshima, Japan: a case study

Three Japanese Black cows housed with 6 other cows exhibited main clinical symptoms of severe hemoglobinuria. Hematological analyses conducted after antibiotic therapy demonstrated severe anemia, and biochemical analyses indicated both severe hemolysis and disruption of hepatic function. One of the three cows died. Based on the above analyses and observation of typical clinical symptoms, a speculative diagnosis of bacillary hemoglobinuria was made, and immediate high-dose antibiotic treatment improved the general conditions of the surviving animals. Blood samples from the other 2 cows were collected sequentially after antibacterial therapy. Clostridium haemolyticum was detected by a nested polymerase chain reaction analysis of the blood samples. The cows were diagnosed with the second recorded occurrence of bacillary hemoglobinuria in Japan.