Biological and environmental factors associated with the detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in Thailand

Elephant endotheliotropic herpesvirus (EEHV) infection is one of the most common diseases in young elephants, causing severe fatal hemorrhagic disease. Subclinical infection was previously described; however, information about the factors associated with virus shedding and reactivation were scarce. To identify the biological and environmental factors related with EEHV detection, blood and oral swab samples were collected from nine captive Asian elephants in Thailand for one year and tested for EEHV presence using real-time PCR. Data including hematological values, management, environmental temperature, and serum cortisol levels were also recorded and analyzed. Results showed that the viral detection frequency ranged from 0–25%. The highest detection frequency was found in the two youngest elephants, aged less than 15 years. Three types of viruses, EEHV1, EEHV4, and EEHV5, were found in this study, which also detected mixed infection in five elephants. Additionally, the study found that sample type, changes in hematological values, management and health issues, and serum cortisol levels were not associated with herpesvirus detection in the elephants. However, EEHV detection percentage was significantly increased in the summer (mid-Feb to mid-May), possibly due to body fitness reduction from food source limitation and low nutrient content. To obtain a broad aspect of EEHV management, long-term EEHV monitoring is highly recommended in every captive elephant herd.     

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine^®, 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

Influence of Salinity and Acidity on Bioavailability of Sludge-Borne Heavy Metals. A Case Study of Bangkok Municipal Sludge

This study was conducted to assess the potential of Bangkoksewage sludge for amendment of saline and acidic soil withrespect to heavy metal mobilization. Experiments were carriedout at six levels of salinity (with electrical conductivityvalues of 2, 4, 8, 19, 31 and 42 dS m^-1) and two levels ofacidity (with pH values of 5 and 6). A pure sludge sample was used as a control system. Subsamples were collected andanalyzed for total and bioavailable heavy metals. Three sets ofsoil-sludge mixture were prepared using different weight ratio(1:1, 4:1 and 10:1) to simulate the conditions in the field withsludge-amended soil. Samples of soil-sludge mixture were incubated for 4 weeks to stabilize organic substances. Themobility and concentration of bioavailable heavy metal invarying soil-sludge mixture ratio with different electricalconductivity (EC) values were evaluated with the use of DTPA(diethylenetriamine pentaacetric acid) extraction techniques.Pure saline soil sample from Sakhon Nakhon (North-eastern partof Thailand) was used of salt-heavy metal interactions on thegermination percentage in soil mixed with sludge and to screencrops or varieties for their tolerances to soil salinity. Seventypes of seeds – mung bean, corn, soybean, tomato, stringbean, sunflower and marigol – were planted in each of the threesoil-sludge mixtures (1:1, 4:1 and 10:1) with different salinitylevels. Seedlings were monitored by counting the number of seeds thatare germinated and seed germination percentage can be used as anindicator for the level of salinity unsuitable for plant growth.     

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region,Thailand

Tropical Animal Health and Production - Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak...     

Improvement of staining method for observation of mitotic chromosomes in octoploid strawberry plants

An important strategy to conduct intentional breeding of octoploid strawberry plants is to recognize the functions of every chromosome. To do so, a methodology must be developed to distinguish chromosomes one by one. We reported the possibility of distinguishing chromosomes using light microscopy when somatic cells of octoploid strawberry plants were stained using ordinary methods with lacto-propionic orcein (LPO). However, karyotype analysis of octoploid strawberry plants required clearer chromosome images. This study obtained clearer chromosome images of octoploid Fragaria × ananassa and F. chiloensis plants. Three staining methods were examined: 60% acetic acid (AA) alone, 1.5% LPO alone, and two-step treatments with 60% AA and 1.5% LPO. Collected root tips of the plants were placed in 2 mM 8-hydroxyquinoline solution for 1 h and were subsequently stored at 4 °C for 15 h. The samples were then fixed in a 3:1 absolute alcohol: glacial acetic acid solution for 40 min, followed by mixture with 1N HCl solutions at room temperature for 2 h and then at 60 °C for 10 min. For separate staining using 60% AA and 1.5% LPO, the root tip was expelled on the glass slide with a drop of each solution for a few minutes to stain the chromosomes. For the two-step staining method, the samples stained with 60% AA were frozen at −80 °C for at least 5 min. The cover slip was removed using a razor blade. Subsequently, the specimens were air-dried and stained with the 1.5% LPO for 3 min. Digital images of chromosomes were obtained using light microscopy. Samples of the two-step staining method produced the clearest chromosome images in both F. × ananassa and F. chiloensis. Furthermore, the greatest color difference between the chromosomes and the cytoplasm was obtained from images of the two-step staining method among the three staining methods. These results demonstrate that the two-step staining method is useful for chromosome counting and karyotype analysis in strawberry plants.     

Effects of dietary protein and lipid content on growth performance and biological indices of iridescent Shark (Pangasius hypophthalmus, Sauvage 1878) fry

Dietary protein and lipid effects on growth, body composition and indices of iridescent Shark Pangasius hypophthalmus (Sauvage 1878) fry were studied using a 4 × 2 factorial design. Triplicate groups of 10 fish per tank, with initial mean weights of 3.54–3.85 g were fed eight isocaloric diets comprising a combination of four protein levels (250, 300, 350 and 400 g kg⁻¹ or 25%, 30%, 35% and 40%) and two lipid levels (60 and 120 g kg⁻¹ or 6% and 12%) respectively. The fish were hand-fed to satiety twice daily for 8 weeks. Specific growth rate (SGR) and feed conversion ratio (FCR) showed significant effects ( P <0.05) with variations in dietary protein and lipid. The highest SGR was observed in fish fed 40% protein/12% lipid diet but this value was not significantly ( P >0.05) different from the fish fed 30% protein/12% lipid diet. The FCR was lowest for the 40/12 diet and differed significantly only with the 25/6, 25/12 and 30/6 treatments respectively. The hepatosomatic index (HSI) was significantly affected by the level of protein, but intraperitoneal fat (IPF) showed significant variation due to dietary lipid level. The HSI significantly ( P <0.05) decreased when dietary protein increased from 25% to 30% but increased marginally thereafter. The IPF values increased with increased dietary lipid but decreased with increased dietary protein. Body protein was positively correlated with dietary protein content; conversely, body lipid content decreased with increase in dietary protein. The results of this experiment indicate the presence of a protein-sparing effect of lipid as fish fed 30% protein/12% lipid diet had growth and feed utilization comparable to those fed 40% protein/12% lipid diet.     

CAPS DNA Marker Labeling Using Primed in Situ Hybridization Technique for Chromosome of Strawberry (Fragaria × ananassa Duch.)

The cleavage amplified polymorphic sequence (CAPS) markers were developed to identify strawberry cultivars from unknown samples. These markers seemed to have a chromosome specificity. However, no reports confirmed it. The present study was conducted to verify the location of a CAPS marker on the chromosomes using a primed in situ (PRINS) technique. A CAPS marker was hybridized with the ‘Sachinoka’ chromosomes using the PRINS technique. The samples were observed under a fluorescence microscope. When the chromosomes were hybridized with the single marker, fluorescent signals were found on two chromosomes. From the results, the CAPS marker was confirmed to have a chromosome specificity by chromosome observation.     

Chromosome observation method at metaphase and pro-metaphase stages in diploid and octoploid strawberries

Chromosome observation is necessary to elucidate the structure, function, organization, and evolution of octoploid strawberry plants’ genes and genomes. However, distinguishing strawberries’ chromosomes from one another using light microscopy is extremely difficult, not only because of their small size and large number, but also because current chromosome observation methods are insufficient. Chromosome preparation and staining using maceration enzymes, acetic acid, and DAPI (4′,6-diamidino-2-phenylindole) were improved for this study to obtain clear images of somatic chromosomes in Fragaria vesca (2n = 14) and Fragaria×ananassa (2n = 56). Collected root tips of octoploid plants were placed in 0.002 M 8-hydroxyquinoline solution for 1 h and stored at 4 °C for 16 h. Subsequently, they were fixed using 3:1 absolute alcohol:glacial acetic acid for 40 min, hydrolyzed in the 1N HCl solution at room temperature for 2 h, macerated using an enzyme solution for 25 min at 42 °C, and stained in 1.5% lacto-propionic orcein solution. On the other hand, in case of DAPI staining, the macerated root tips of octoploid plants were soaked in 60% acetic acid for 5 min before staining. Clear digital images of F. vesca and F.×ananassa were obtained using light and fluorescent microscopy. Their respective 14 and 56 chromosomes were counted. Fluorescent microscopy yielded clear chromosome images at the pro-metaphase in F. vesca and F.×ananassa. This chromosome observation method alleviates the difficulties that have heretofore hindered chromosome analyses of strawberry plants.