A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

A simple strip-test kit for white spot syndrome virus (WSSV) detection was developed using monoclonal antibody W29 (against the VP28 structural protein) conjugated with colloidal gold as the detector antibody. A rabbit anti-recombinant VP28F118 (rVP28) protein antibody in combination with a W28 monoclonal antibody was used as the capture complex at the test line (T), and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). For evidence, the ready-to-use strip was kept in a plastic case and stored in a desiccated plastic bag. A sample volume of 100 microl gill homogenate in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing WSSV, the virus bound to the monoclonal antibody conjugated with colloidal gold and the resulting complex was captured by the antibodies at T to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across T to be captured by the GAM and formed a band at C. In samples without WSSV or with WSSV below the limit of detection of the kit, only the band at C was seen. This method was 4 times less sensitive than dot blotting, and about 2 000 000 times less sensitive than 1-step PCR. Nonetheless, it could be used to screen individual shrimp or pooled shrimp samples to confirm high levels of WSSV infection or WSSV disease outbreaks. The beneficial features of this kit are that simple, convenient and quick results can be obtained without the requirement of sophisticated tools or special skills.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

A novel modified culture medium for enhancing redifferentiation of chondrocytes for cartilage tissue engineering applications

The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days. Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83–2.75, 5.00–7.50, and 1.00–1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.     

Differences in susceptibility of palaemonid shrimp species to yellow head virus (YHV) infection

Five species of palaemonid shrimp, Macrobrachium rosenbergii, M. lanchesteri, M. sintangense, Palaemon styliferus and P. serrifer, were collected from Penaeus monodon farming areas in Thailand. Some of each species were artificially infected with yellow head virus (YHV) by injection and then monitored by RT-PCR and by immunohistochemistry using monoclonal antibodies specific to 116 kDa, 64 kDa, and 20 kDa proteins of YHV. Natural YHV infections were not detected in any of the shrimp examined. In YHV injection experiments, a high proportion of P. serrifer, P. styliferus and M. sintangense exhibited mild to moderate YHV infections at 3 d post-injection. The severity of infection was reduced in shrimp that survived to 10 and 30 d post-injection. Using immunohistochemistry and RT-PCR, a small proportion of M. lanchesteri showed very mild YHV infections at Day 3 but no infections at Days 10 and 30. No YHV infections resulted in M. rosenbergii. The evidence suggested that M. sintangense, P. styliferus and P. serrifer are susceptible to YHV and carry it for some time. In contrast, M, rosenbergii and M. lanchesteri appear to resist YHV infection and eliminate YHV efficiently. Because they display a range of responses to YHV, palaemonid shrimp may serve as a good model for studying YHV defense mechanisms in shrimp.     

Inter-regional mating compatibility among Bactrocera dorsalis populations in Thailand (Diptera,Tephritidae)

Mating compatibility among recently colonized (wildish) populations of Bactrocera dorsalis (Hendel) from different geographic origins in Thailand was assessed through inter-regional mating tests. Outdoor octagonal nylon screen field cages containing single potted mango trees (Mangifera indica L.) were used. Sexual compatibility was determined using the index of sexual isolation (ISI), the male relative performance index (MRPI), and the female relative performance index (FRPI). The ISI values indicated that the northern population of Bactrocera dorsalis from Chiang Mai province was sexually compatible with the southern population of Bactrocera dorsalis (previously Bactrocera papayae) from Nakhon Si Thammarat province. The MRPI values showed that the northern males had a slightly higher tendency to mate than southern males, while the FRPI data reflected that females of both origins participated equally in matings. In all combinations there were no differences between homotypic and heterotypic couples in mating latency. Southern males tended to mate first with southern females, followed by northern males mating with northern females, while the latest matings involved heterotypic couples, in particular northern males mating with southern females. Overall, more couples were collected from higher parts of the field cage and the upper tree canopy, while there were no differences between the origins of flies in terms of elevation of couples within the cage. Laboratory assessments of fecundity showed no differences in the average number of eggs resulting from inter-regional crosses. Development of immature stages was also equal in the two hybrid crosses, with no differences found in the number of pupae produced, percentage pupal recovery, and percent adult emergence. The practical implication of this study is that colony of Bactrocera dorsalis derived from any northern or southern region of Thailand can potentially be used in sterile insect technique programs against this pest.     

Production of monoclonal antibodies for detection of Vibrio harveyi

Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10(4) CFU ml(-1) using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits.     

Neutralizing Dengue Antibody in Pregnant Thai Women and Cord Blood

Background

The WHO ‘Global Strategy for Dengue Prevention and Control, 2012–2020’ addresses the growing need for the treatment of dengue, and targets a 25% reduction in morbidity and 50% in mortality (using 2010 estimates as baseline). Achieving these goals requires future dengue prevention strategies that will employ both potential vaccines and sustainable vector-control measures. Maternally transferred dengue antibody is an important factor in determining the optimal age for dengue vaccination.

Objectives

To estimate the seroprevalence of dengue antibodies among mothers living in an area of high endemicity – Ban Pong, Ratchaburi Province – and to assess maternal dengue antibodies transferred to cord blood.

Materials & Methods

A cross-sectional study was conducted with 141 pregnant women who delivered at Ban Pong Hospital, Ratchaburi, Thailand. Maternal-cord paired sera were tested for dengue neutralizing (NT) antibody by PRNT₅₀ assay. A ratio of ≥ 1:10 NT titer to dengue serotype was considered seropositive.

Results

Most mothers (137/141, 97.2%) had NT antibodies to at least one dengue serotype in their sera. At birth, the proportion of cord sera with NT antibodies to DEN-1, DEN-2, DEN-3, and DEN-4, were high and similar to the sera of their mothers, at 93.6%, 97.2%, 97.9%, and 92.2%, respectively. The dengue geometric mean titers (GMT) in cord blood were significantly higher than the maternal antibodies (p<0.001): highest in DEN-2, followed by DEN-3, and then DEN-1. The GMT of DEN-4 was the lowest among all four serotypes.

Conclusions

Dengue infection is highly prevalent among pregnant women in this dengue-endemic area. Most of the cord blood had transferred dengue antibodies, which may have an impact on the disease burden in this population.     

Controlled ecdysteroid accumulation in eggs of the silkworm,Bombyx mori,by an imidazole compound (KK-42), and embryogenesis in these eggs

An imidazole compound (KK-42), a potent inhibitor of ecdysone synthesis, was applied to the female pharate adult of the silkworm, Bombyx mori, to control ecdysteroid accumulation in developing ovaries and mature eggs. KK-42 applied on day 2 or later completely suppressed an increase in ecdysteroid content in developing ovaries. The inhibitory action of KK-42 was restricted to vitellogenic follicles, i.e., those in which active ecdysteroid synthesis is occurring. Ecdysteroid content in the mature eggs of moths remained at the level accumulated in ovaries before KK-42 application. Thus, KK-42 was shown to be a novel agent to suppress the ecdysteroid accumulation in eggs. Eggs containing different amounts of ecdysteroids showed different levels of embryonic development. About 80% of the eggs which contained less than 10 ng free ecdysteroids/g eggs were not fertilized. More than 80% of the eggs containing less than 40 ng/g eggs of free ecdysteroids initiated embryogenesis but failed to hatch. Larvae hatched from almost all eggs which accumulated free ecdysteroids of more than 150 ng/g. Thus, maternal ecdysteroids appear to be required at different titers for fertilization, embryogenesis, and hatching of the silkworm larvae. © 1994 Wiley-Liss, Inc.     

Molecular phylogeny of Asian termites (Isoptera) of the families Termitidae and Rhinotermitidae based on mitochondrial COII sequences

The families Termitidae and Rhinotermitidae are the most evolved and diverse groups of the social insects, termites (Order Isoptera), showing elaborated morphology and complex behavior. Molecular phylogeny of termites with the emphasis on these families was examined by Bayesian and maximum-likelihood analyses based on DNA sequence of mitochondrial cytochrome oxidase II (COII) gene of 31 genera sampled in Asia (mainly Thailand and Japan) along with those reported previously. Termitidae was monophyletic and originated from within polyphyletic Rhinotermitidae. Among the four subfamilies of Termitidae, Macrotermitinae was monophyletic suggesting a single common origin of fungus-growing habit characteristic for this subfamily, and was placed in the basal position in the family. A group consisting of other subfamilies Termitinae and Nasutitermitinae, though some important groups were still untouched, was the most apical but neither Termitinae nor Nasutitermitinae formed a monophyletic lineage. It was implied that, as defense systems of the soldier castes, the appearance of snapping mandibles has occurred at a single event, but the development of nasus for chemical secretion has probably not. Our tree provides some evidence concerning contradictions in the previously proposed phylogeny of termites.