Epizootology and experimental infection of Yokose virus in bats

To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.     

A toxicity bioassay study concerning the effect of un-ionized ammonia on the mucus cells response originating from the gills of zebrafish Danio rerio

Ammonia is one of the most common and pervasive pollutants in the aquatic habitat affecting the health of aquatic animals. It affects the number and morphology of mucus producing cells, resulting in excessive and irregular secretion and alterations in quantity of mucus, which can lead to complications in fish health. A toxicity test on the mucus cells localized on the gills (primary lamellae) and inner operculum epithelial lining of zebrafish Danio rerio at different periods of exposure (24, 48 and 72 h) followed by recovery periods was studied using histopathology, scanning electron microscopy and anterior gradient 2 homologue (agr2) gene expression techniques. Fish samples subjected to low, medium and high external toxic ammonia concentrations (L: 17.21 mg/l, M: 25.81 mg/l and H: 38.91 mg/l NH₃-N) were observed to display a higher mucus layer production and active secretion compared with the control. Gill cellular alterations were more severe at 48 and 72 h. A high expression of agr2 was detected at 48 h (L and M) recovery periods and a (H) exposure and recovery period indicating an increase in quantity in newly proliferated alcian blue stained mucus cells and excessive secretion observed by histopathology. Such expression decreased at 72 h resulting in a decrease in mucus cell density and secretion.     

Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20β-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen–antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml^?1. The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).     

Ultra highly sensitive method for detecting Flavobacterium psychrophilum using high‐gradient immunomagnetic separation with a polymerase chain reaction

We attempted to develop an ultrahigh sensitive method for detecting Flavobacterium psychrophilum using high‐gradient immunomagnetic separation (HGIMS) with a polymerase chain reaction (PCR). HGIMS is a magnetic separation method in which the magnetic force is strengthened by introducing a magnetic gradient between the magnetic filter and nearby column. Because immunomagnetic beads specifically react with target cells, target cells are collected efficiently. Accumulated beads are released from the filter by removing the external magnetic force. After concentrating the samples using the HGIMS system, DNA was extracted from the samples, and PCR was applied to detect F. psychrophilum. Our primers did not react with reference bacteria and reacted specifically with F. psychrophilum. The detection sensitivity using the HGIMS system was higher than that of the method without the HGIMS system, and the total assay time, including sample preparation, was <3.5 h. PCR products of the expected size were obtained from samples of concentrated 4 × 10^?1 to 4 × 10³ cfu mL^?1F. psychrophilum more than 80% of the time using the HGIMS system. Furthermore, our proposed method could be useful for the specific detection of F. psychrophilum from actual samples. Our proposed method is suitable for the highly sensitive detection of F. psychrophilum.     

Atmospheric moisture influences on conidia development in Podosphaera xanthii through host-plant morphological responses

This study investigated the effects of atmospheric moisture on conidia development in cucurbit powdery mildew fungus (Podosphaera xanthii) through host-plant responses. Cucumber (Cucumis sativus L.) seedlings were grown under a high vapour pressure deficit (VPD) (3.8 kPa) or a low VPD (0.4 kPa). When the cotyledons had expanded, spores of P. xanthii were inoculated onto the adaxial surface. Inoculated seedlings for both treatments were then placed in a growth chamber maintained at a VPD of 2.1 kPa. The density of visible P. xanthii colonies on the high-VPD-acclimated cotyledons was 0.46 to 0.85 times that of the low-VPD-acclimated cotyledons 7 days after inoculation. It is likely the post-germination behaviour of P. xanthii such as the infection and consequent hyphal development was affected because spore germination did not differ between the treatments. The percentage of adaxial epidermal leaf cells with haustoria was also lower in the seedlings which had acclimated to a higher VPD. The high-VPD-acclimated cotyledons were thicker and had greater dry mass per area. The water potential of cotyledons did not differ between the treatments, although the stomatal conductance of high-VPD-acclimated cotyledons was lower than that of low-VPD-acclimated cotyledons. From these results, we conclude that the inhibition of P. xanthii conidia development on high-VPD-acclimated cotyledons was mainly caused by changes in leaf morphological properties.     

Assessment for predicting parturition in mares based on prepartum temperature changes using a digital rectal thermometer and microchip transponder thermometry device

The purpose of this study was to observe the changes in body temperature before parturition using a wireless temperature monitoring device (WTMD) and to evaluate the usefulness of body temperature measurements using a digital rectal thermometer (DRT) and a microchip transponder thermometry device (MTTD) for predicting parturition in mares. The body temperatures using a WTMD at 0 hr and -1 hr were significantly different from those at the same time on Days 1-5 (P<0.01). The temperature differences between the morning of Day 0 and at -3 hr, -2 hr, -1 hr and 0 hr using the DRT and MTTD showed a significant drop compared with the temperature differences between the morning and evening of Days 1-5 (P<0.05). Furthermore, when the cutoff value of the temperature differences between the morning and other times was set to ≤0, the sensitivities of the DRT and MTTD in the evening of Day 0 and at -3 hr were 43% and 100% and 71% and 86%, respectively. The results suggested that monitoring the body temperature differences between morning and within 3 hr before the time of parturition is a valuable method for predicting parturition in mares. Conversely, this method would be more useful in predicting parturition when used in combination with other observations such as the mammary gland size and waxing of the teat ends because it has nearly a 20% probability of false-positive results prior to the day of parturition.     

Application of truncated nucleocapsid protein (N) for serotyping ELISA of murinae-associated hantavirus infection in rats

Truncated recombinant nucleocapsid proteins (trNs) that lack N-terminally located cross-reactive epitopes of four Murinae rodent-associated hantaviruses, Seoul virus (SEOV), Thailand virus, Hantaan virus (HTNV) and Dobrava-Belgrade virus, were produced by using a baculovirus expression system. ELISA with the trNs as antigens enabled serotyping of immune sera from rats experimentally inoculated with the corresponding hantaviruses with cut-off OD values of 60% of those of whole N of HTNV. The trN-based ELISA could serotype 12 out of 13 sera obtained from wild rodents (Rattus norvegicus) naturally infected with SEOV using the 60% cut-off value. These results indicate that screening with whole N followed by serotyping with trNs using a cut-off OD value of 60% of that of whole N is a useful method for serological surveillance of Murinae-associated hantavirus infection among rodents.     

Peripheral lymphocyte subsets as a prognostic indicator of mortality and morbidity in healthy dogs

To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3(+), CD4(+), CD8(+) and CD21(+) cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4(+) and the CD4(+):CD8(+) ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3(+) lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity.     

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.