Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Conservation of Pteropus livingstonii based on roost site habitat characteristics on Anjouan and Moheli, Comoros islands

Livingstone's fruit bats, Pteropus livingstonii, (Pteropodidae: Chiroptera) are endemic to only two islands, Moheli and Anjouan of the Comoros Islands in the West Indian Ocean. With an estimated population of less than 2000 individuals, P. livingstonii is listed as Critically Endangered on the International Union for the Conservation of Nature's Red List. Yet the habitat characteristics of these giant fruit bats, or flying foxes, are poorly known. I conducted field research during 5 months in 1998 focused on finding new roost sites, measuring characteristics of both roost sites and roost trees at new as well as previously identified roost sites, and recording the same characteristics for a set of absence sites. I identified six new roost sites allowing me to measure the characteristics of 15 known roost sites and seven absence sites. The study spanned both dry and rainy seasons and I found a significant increase in the number of individuals at roost sites during the rainy season. Bat roost sites were associated with presence of water, southeast aspect, steep slopes, and presence of several native tree species. Roost habitat was characterized based on biotic, abiotic and human factors. This research on habitat characteristics is contributing to the development of a conservation action plan for the Livingstone's fruit bats. However, further research on dry season roost sites and distribution as well as feeding sites is necessary in order to better understand their seasonal habitat requirements and therefore ensure the conservation of P. livingstonii.     

A Generalized Two-Dimensional Gaussian Model of Disease Foci of Head Blight of Wheat Caused by Gibberella zeae

ABSTRACT A generalized two-dimensional Gaussian model is proposed to describe disease foci of head blight of wheat in plots (100 to 2,500 m(2)) originating from small areas (1 to 16 m(2)) inoculated with Gibberella zeae-colonized corn kernels. These anisotropic, asymmetrical foci arose from ascospores produced in perithecia. The model is Z = exp[-(AX(2) + BY(2) + CXY + DX + EY + F)], in which Z = the incidence of seed or spikelet infection at point (X,Y) located in the plot, exp = the exponential function, X = the abscissa or spatial coordinate of the point along a given axis (approximately parallel to the average wind vector during the period of spore release in these experiments), Y = the ordinate or spatial coordinate of the point along the axis perpendicular to the X axis (approximately perpendicular to the wind direction in these experiments), A and B = the quadratic coefficients of the second-order polynomial AX(2) + BY(2) + CXY + DX + EY + F, C = the bilinear coefficient, D and E = the linear coefficients, and exp(-F) = the incidence of seed or spikelet infection at the focus peak in which X = 0 and Y = 0. The generalized two-dimensional Gaussian model was tested on data from a circular or isotropic focus, an elliptical or anisotropic focus with two axes of symmetry, and two anisotropic foci with one and zero axis of symmetry. Its goodness-of-fit (r(2) and adjusted r(2)) was compared with the inverse power, modified inverse power, exponential, and classical Gaussian models. Submodels using only the linear terms, only the quadratic terms, or combinations selected from stepwise regression procedures using various probabilities to enter and to stay and a procedure maximizing the adjusted r (2) were also considered. Spatial analysis of the residuals was performed using Geary's c coefficient at the first distance class. For the circular and elliptical foci, our model provided a fit similar to the modified inverse power and exponential models. However, for anisotropic foci with one or zero axis of symmetry arising from ascospores influenced by wind direction, the generalized two-dimensional Gaussian model provided a better fit. For these anisotropic foci, the linear term X but not the quadratic term X(2) was generally retained in the model, indicating an exponential gradient in the direction parallel to the wind. In all models, the quadratic term Y(2) was retained, along with Y in some cases, indicating that the gradient in the direction roughly perpendicular to the wind was Gaussian or Gaussian-exponential in shape. The bilinear term XY provided an indication of the orientation of the focus in relation to the axes of the sampling grid. This model has the versatility and parameters (quadratic, bilinear, and linear) to better describe the anisotropy of foci from wind-dispersed spores.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.     

On farm observations to increase genetic gain in breeding schemes for village poultry production – A simulation study

To improve genetic gain of breeding programs for village poultry production, breeding schemes with observations obtained in village production systems using individual (VIO) and group recording (VGO) were examined under different levels of genotype-by-environment-interactions (GxE). GxE was modeled by varying the correlation between traits measured in the breeding station and village environments for bodyweight (r_{g_BW}) and egg production (r_{g_EP}). Relative and absolute genetic gains obtained from VIO and VGO were used for comparison between the schemes. Results showed that village observations significantly improved genetic gains compared to the scheme without birds tested in the village. The improvement was only slightly larger with individual observations than with group observations. Higher r_{g_BW} and r_{g_EP} led to lower relative genetic gain, but a higher absolute gain of VIO and VGO. It is recommended to apply a breeding scheme using group recording of village performance when strong GxE in breeding for village poultry is expected.     

The role of dietary methionine concentrations on growth,metabolism and N‐retention in cobia (Rachycentron canadum) at elevated water temperatures

This study determined impacts of dietary methionine concentrations at two temperatures on growth, feeding efficiency and N‐metabolites in juvenile cobia. Methionine concentrations of the experimental diets were deficient (M9; 9 g/kg), sufficient (M12; 12 g/kg) and surplus (M16, 16 g/kg). Water temperature was normal (30°C) or elevated (34°C). Twenty cobia in triplicate tanks were fed the experimental diets for 6 weeks. Both methionine and temperature affected cobia's growth and feeding efficiency. Cobia fed M9 performed lower than the fish fed M12 and M16 diets. Additionally, cobia reared at 34°C performed poorer than at 30°C, probably due to lower voluntary feed intake in the fish reared at 34°C. Protein efficiency ratio and protein productive value in cobia fed M9 diet were less than M12 or M16 diets. This was confirmed with the improved retentions of indispensable amino acids (AAs). No interactions between methionine and temperature were observed in growth and protein accretion. At 30°C, CF improved, while HSI and VSI declined upon methionine supplementation levels. Of which an interaction between temperature and methionine was present. Plasma, muscle and liver free AA and N‐metabolites were affected by methionine and temperature. Furthermore, temperature affected cobia's lipid class composition, resulting in increased phospholipids and cholesterol at 34°C.     

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.     

Fatigue of structural plywood under cyclic shear through thickness I: fatigue process and failure criterion based on strain energy

The fatigue behavior of plywood specimens under shear through thickness was examined on the basis of strain energy to obtain common empirical equations for the fatigue process and failure criterion under various loading conditions. Specimens were cut from commercial plywood panels of 9-mm thickness. Loading conditions were set as follows: a square waveform at a loading frequency of 0.5 Hz, a triangular waveform at 0.5 Hz, and a triangular waveform at 5.0 Hz. Peak stress applied was determined to be 0.5, 0.7, and 0.9 of static strength, that is, stress levels of 0.5, 0.7, and 0.9. The stress-strain relationships were measured throughout the fatigue test, and the strain energy was obtained at each loading cycle. Loading conditions apparently affected the relationship between stress level and fatigue life. On the other hand, the relationship between mean strain energy per cycle and fatigue life was found to be independent of loading conditions. Mean strain energy per cycle obtained as the fatigue limit was 5.85 kJ/m³ per cycle. Assuming that the accumulation of strain energy is a fatigue indicator, the fatigue process and failure criterion for the plywood specimens under the three loading conditions were commonly expressed by the relationship between cumulative strain energy and loading cycles.     

Predicting summer fin whale distribution in the Pelagos Sanctuary (north-western Mediterranean Sea) to identify dynamic whale–vessel collision risk areas

1. Mediterranean fin whales aggregating in the Pelagos Sanctuary in summer to feed are exposed to vessel collision risk, particularly from high-speed ferries.
2. This study developed models to predict summer fin whale distribution using a generalized additive model (GAM) and MaxEnt, with the aim of providing a tool to identify potential high whale–ferry collision risk areas along ferry routes within the Pelagos Sanctuary during summertime.
3. Models were trained using sightings data collected in the summer months of 2009–2018 on board ferries crossing the central area of the Pelagos Sanctuary. Environmental predictors were bathymetry and mean sea surface chlorophyll concentration of the annual spring bloom period.
4. The predictive ability of GAM and MaxEnt was assessed using existing knowledge of summer fin whale distribution in the region. GAM (deviance explained = 20.2%) predictions matched documented distributions more closely than that of MaxEnt, with highest predicted fin whale occurrence in deep offshore waters (>2000 m) encompassing the central north-western and western regions, and in the south-eastern region, consistent with known fin whale habitats within the Pelagos Sanctuary. Inter-annual variability was evident, influencing collision risk areas.
5. Collision risk was estimated as a function of the overlap between the predicted probability of fin whale occurrence and ferry density estimated from Automated Identification System data. Ferry routes that cross the northern and eastern regions of the Pelagos Sanctuary presented relatively higher collision risk.
6. Areas with changes in risk intensity between the years were temporally and spatially dynamic: some appeared intermittently throughout the study period while others persisted over consecutive years or recurred in different years.
7. Due to the vastness of the Pelagos Sanctuary, vessel speed reduction maybe a more practical measure to manage collision risk than re-routing shipping lanes. A combination of Seasonal Management Areas and Dynamic Management Areas approaches could be adopted for high-risk areas.