Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.     

Relationship between transportation stress and polymorphonuclear cell functions of bottlenose dolphins, Tursiops truncatus

Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.     

Colonic mast cell infiltration in rats with TNBS-induced visceral hypersensitivity

Colonic mucosal mast cells are implicated in the pathogenesis of visceral hypersensitivity associated with irritable bowel syndromes. This study was designed to investigate the roles of mucosal mast cells in development of an experimental visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. TNBS, when injected into the proximal colon through laparotomy, produced a significant decrease in pain threshold of the distal colon to mechanical distention, indicating a visceral hypersensitivity. In the proximal colon that was directly insulted by TNBS, mucosal necrosis and extensive inflammatory cell infiltration were observed with concomitant increase in tissue myeloperoxide (MPO) activity. In the distal colon where distention stimuli were applied, the number of mucosal mast cells significantly increased following TNBS treatment, although neither mucosal injury nor increase in tissue MPO activity was observed. In an organ culture, spontaneous release of a mucosal mast cell-specific protease (RMCP-2) from the distal colon tissue of TNBS-treated rats was significantly larger than that of sham animals. Furthermore, TNBS-induced visceral hypersensitivity was significantly suppressed by subcutaneous pretreatment with a mast cell stabilizer doxantrazole in a dose-dependent manner. These findings suggest that prominent colonic mast cell infiltration associated with an enhanced spontaneous mediator release is responsible, at least partly, for development of visceral hypersensitivity induced by TNBS in rats.     

Prevalence of tick-borne Rickettsia and Ehrlichia in Ixodes persulcatus and Ixodes ovatus in Tokachi district, Eastern Hokkaido, Japan

DNA from 111 ticks collected by flagging in Tokachi district, Eastern Hokkaido, Japan were examined for infection with Rickettsia and Ehrlichia, by PCR and sequencing methodology. For Rickettsia, analysis of the partial sequence of the citrate synthase gene was successfully performed on 11 DNA samples from I. persulcatus, and 7 of them showed 99.8% identical with Rickettsia helvetica while the other 4 showed 99.8% identical with ;Candidatus Rickettsia tarasevichiae'. For Ehrlichia, a partial sequence of the 16S rRNA gene detected from I. persulcatus was 100% identical with that from Ehrlichia muris, and another DNA sample from I. ovatus showed 99.8% identical with Ehrlichia species detected from I. ovatus. The results suggest that the pathogens detected here might be distributed in this area.     

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido,Japan

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006–2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.     

Heparin inhibits plaque formation by cell-free Marek's disease viruses in vitro

The mechanisms of Marek's disease virus (MDV) entry to host cells have not yet been analyzed. Heparan sulfate (HS) on the cell surface serves as a receptor for several herpesviruses in mammalian species. In this study, we demonstrated that plaque formation by cell-free MDV is inhibited by the addition of soluble heparin to the cell culture. Moreover, pretreatment of susceptible cells, chicken embryo fibroblasts, with heparinase, partially reduced infectivity of the cell-free MDV. From these results, it was suggested that the MDV entry, at least in the case of cell-free MDV, is dependent on the presence of cell surface glycosaminoglycans, principally HS.