Experimental studies on the portal of entry of White spot syndrome virus in Penaeus vannamei

Over the last decades the rapid expansion of global shrimp aquaculture industry was accompanied by an increased incidence of infectious diseases with white spot syndrome (WSS) as the most important one. Despite many attempts to prevent WSS, effective control measures have not been found yet. Finding the portal of entry of WSSV will help in understanding the viral transmission in between shrimp. Together with the investigation of the factors determining viral transmission, it is an essential step for the development of WSSV control measures

Per os infectivity of white spot syndrome virus (WSSV) in white-legged shrimp (Litopenaeus vannamei) and role of peritrophic membrane

As earlier observations on peroral infectivity of WSSV in white-legged shrimp are conflicting, here, a standardized peroral intubation technique was used to examine (i) the role of the physical composition of the viral inoculum and (ii) the barrier function of the PM. In a first experiment, the infectivity of a WSSV stock was compared by determining the SID50 by intramuscular injection, peroral inoculation or via feeding. The following titers were obtained: 108.77 SID50/g by intramuscular injection, 10(1.23) SID50/g by peroral inoculation and 100.73 SID50/g by feeding. These results demonstrated that 10(7.54)-10(8.03) infectious virus is needed to infect shrimp by peroral inoculation and via feeding. Next, it was examined if damage of the PM may increase the susceptibility for WSSV by peroral route. The infectivity of a virus stock was tested upon peroral inoculation of shrimp with and without removal of the PM and compared with the infectivity upon intramuscular inoculation. The virus titers obtained upon intramuscular injection and peroral inoculation of shrimp with and without PM were 10(8.63), 10(1.13) and 10(1.53) SID50/mL, respectively. This experiment confirmed the need of 10(7.1)-10(7.5) infectious virus to infect shrimp via peroral route and showed that the removal of the PM slightly but not significantly (p > 0.05) facilitated the infection of shrimp. This study indicated that WSSV contaminated feed is poorly infectious via peroral route, whereas it is highly infectious when injected into shrimp. The PM plays a minor role as internal barrier of shrimp against WSSV infection

Antifungal Susceptibility of Dermatophytes Isolated From Cutaneous Fungal Infections: The Vietnamese Experience

AIM: Evaluate the resistance of dermatophytes to systemic antifungal drugs in the Vietnamese population. METHODS: We enrolled 101 patients with cutaneous dermatophytosis at the Dermato-Venereology hospital in HCMC from August 2016 to March 2017. All the specimens were subjected to direct examination (10% KOH mount) and culture on Sabouraud dextrose agar. In vitro antifungal sensitivity testing was done on species isolated from a culture with broth microdilution method. RESULTS: Direct microscopy was positive for dermatophytes in all patients. However this pathogen was found in fungal cultures in only 61.38% of patients. The main causative agent isolated was Trichophyton spp. (90.3%), followed by Microsporum spp. (8%) and Epidermophyton spp. (1.7%). Trichophyton spp. Has shown resistance to fluconazole, griseofulvin, ketoconazole, and itraconazole in 92.9%, 46.4%, 5.4% and 1.8% of strains, respectively. All Microsporum spp. Strains were found resistant to fluconazole and griseofulvin while resistance to ketoconazole was demonstrated in only 20% of strains and none of them was resistant to itraconazole. Epidermophyton spp strains were all resistant to fluconazole, griseofulvin, ketoconazole while none of them was resistant to itraconazole. CONCLUSION: Based upon our results, Itraconazole shows the greatest probability of efficacy in the treatment of cutaneous dermatophytosis in Vietnamese patients

Susceptibility of juvenile Macrobrachium rosenbergii to different doses of high and low virulence strains of white spot syndrome virus (WSSV)

As some literature on the susceptibility of different life stages of Macrobrachium rosenbergii to white spot syndrome virus (WSSV) is conflicting, the pathogenesis, infectivity and pathogenicity of 2 WSSV strains (Thai-1 and Viet) were investigated here in juveniles using conditions standardized for Penaeus vannamei. As with P. vannamei, juvenile M. rosenbergii (2 to 5 g) injected with a low dose of WSSV-Thai-1 or a high dose of WSSV-Viet developed comparable clinical pathology and numbers of infected cells within 1 to 2 d post-infection. In contrast, a low dose of WSSV-Viet capable of causing mortality in P. vannamei resulted in no detectable infection in M. rosenbergii. Mean prawn infectious dose 50% endpoints (PID50 ml(-1)) determined in M. rosenbergii were in the order of 100-fold higher for WSSV-Thai-1 (10(5.3 +/- 0.4) PID50 ml(-1)) than for WSSV-Viet (10(3.2 +/- 0.2) PID50 ml(-1)), with each of these being about 20-fold and 400-fold lower, respectively, than found previously in P. vannamei. The median lethal dose (LD50 ml(-1)) determined in M. rosenbergii was also far higher (similar to 1000-fold) for WSSV-Thai-1 (10(5.4 +/- 0.4) LD50 ml(-1)) than for WSSV-Viet (10(2.3 +/- 0.3) LD50 ml(-1)). Based on these data, it is clear that juvenile M. rosenbergii are susceptible to WSSV infection, disease and mortality. In comparison to P. vannamei, however, juvenile M. rosenbergii appear more capable of resisting infection and disease, particularly in the case of a WSSV strain with lower apparent virulence

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Eye extract improves cell migration out of lymphoid organ explants of L-vannamei and viability of the primary cell cultures

Since no cell line from shrimp has been established up till now, an optimization of the primary cell culture protocol is necessary. In this context, the effect of extracts (supernatant of a 1:50 (w/v) suspension) from different shrimp organs (muscle, brain, ganglia, eyestalk, ovary, and eye) on the performance of primary lymphoid cell cultures was evaluated. Ten percent of eye extract and 3% of ovary extract enhanced maximally the migration and survival of cells of the lymphoid organ of Litopenaeus vannamei significantly at 48, 96, and 144 h post seeding. Extracts from the eyestalk (10%), muscle (10%), and brain (1%) significantly promoted the migration and survival of cells at 48 and 96 h post seeding but not anymore at 144 h post seeding. In conclusion, it may be advised to add 10% of eye extract or 3% of ovary extract to cells for the maximal health of primary cell cultures from the lymphoid organ of L. vannamei

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35gL(-1) were subjected to salinities of 50gL(-1), 35gL(-1), 20gL(-1), 10gL(-1) and 7gL(-1) or 5gL(-1) and simultaneously exposed to 10(5.5)SID(50) mL(-1) of WSSV for 5h, after which the salinity was brought back to 35gL(-1). Shrimp that were transferred from 35gL(-1) to 50gL(-1), 35gL(-1) and 20gL(-1) did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35gL(-1) to 10gL(-1) and lower. The mortality in shrimp, subjected to a salinity change to 10gL(-1), 7gL(-1) and 5gL(-1), was 6.7%, 46.7% and 53.3%, respectively (P<0.05)(.) For testing the effect of moulting, shrimp in early premoult, moulting and post-moult were immersed in sea water containing 10(5.5)SID(50) mL(-1) of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post-moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P<0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10gL(-1) and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures