Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Gray (Oreochromis niloticus x O. aureus) and Red (Oreochromis spp.) Tilapia Show Equal Susceptibility and Proinflammatory Cytokine Responses to Experimental Tilapia Lake Virus Infection.

Tilapia is the second most farmed fish species after carp in the world. However, the production has come under threat due to emerging diseases such as tilapia lake virus (TiLV) that causes massive mortalities with high economic losses. It is largely unknown whether different tilapia strains are equally susceptible to TiLV infection. In the present study we compared the susceptibility of gray (Oreochromis niloticus x O. aureus) and red tilapia (Oreochromis spp.) to experimental TiLV infection. Virus was injected intraperitoneally at a concentration of 104 TCID50/mL. Our findings show that gray tilapia had a lower mortality, 86.44%, but statistically not significantly different (p = 0.068) from red tilapia (100%). The duration of the mortality period from onset to cessation was similar for the two species, starting at 2-3 days post challenge (dpc) with a median at 10-11 dpi and ending on 20-22 dpi. In addition, there was no difference between species in mean viral loads in brain, liver and headkidney from fish collected soon after death. As for host response, expression levels of IL-1ß and TNFα were equally high in brain and headkidney samples while levels in liver samples were low for both red and gray tilapia, which coincides with lower viral loads in liver compared to brain and headkidney for both species. We find that red and gray tilapia were equally susceptible to TiLV infection with similar post challenge mortality levels, equal virus concentration in target organs and similar proinflammatory cytokine responses in target and lymphoid organs at time of death. Nonetheless, we advocate that the search for less susceptible tilapia strains should continue with the view to reduce losses from TiLV infection in aquaculture.

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda.

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A. hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A. hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.

Seroprevalence of hepatitis E in swine abattoir workers.

BACKGROUND: Hepatitis E (HE) caused by Hepatitis E virus (HEV) is an emerging global public health threat. It has been identified as potentially zoonotic and swine act as main reservoirs. OBJECTIVES: The objective of this study was to determine the seroprevalence and risk factors associated with HEV in swine abattoir workers. METHODS: This was a cross-sectional study where 45 workers were sampled (N=50), serum collected and tested for presence of anti HEV IgM using ELISA. RESULTS: A seroprevalence of 13.3% was obtained with the highest 50% among slaughterers and the lowest amongst sanitary cleaner, cloth cleaners and inspector. Those in direct contact with live pigs, their carcasses and tissues were at a higher risk compared to those in indirect contact. Seroprevalence was seen to increase with age, with the highest rate among those above 24 years. CONCLUSION: There is silent HE virus infection in abattoir workers at Wambizi as reflected by presence anti HEV IgM in 13% of the tested serum. However, no single case of HE has ever been reported in swine abattoir workers or general population in Kampala city. This silent maintenance of HEV infection amongst swine abattoir workers is an occupational risk that could challenge public health systems.