Isolation of Streptococcus cuniculi from corneal lesion in laboratory-raised mice.

Corneal lesions appearing as white mass beneath intact epithelium, with ocular discharge in one mouse, was observed in a batch of laboratory-raised BALB/c mice (n=9 of 56). The affected mice remained active, well-groomed and had normal appetite. Isolates recovered from swab cultures of the external and internal contents of the eye had partial 16S rRNA gene sequence of 99.1% similarity to Streptococcus cuniculi. No previous report of S. cuniculi infection in laboratory rodents has been presented. The isolate was susceptible to all antibiotics tested. We suggest S. cuniculi is an opportunistic bacteria in laboratory mice but are uncertain of its source. Our findings revealed that S. cuniculi is able to colonize laboratory mice and should be considered when mice present with eye lesion or ocular discharge.

Oral immunization of a non-recombinant Lactococcus lactis surface displaying influenza hemagglutinin 1 (HA1) induces mucosal immunity in mice.

Mucosal immunization of influenza vaccine is potentially an effective approach for the prevention and control of influenza. The objective of the present study was to evaluate the ability of oral immunization with a non-recombinant Lactococcus lactis displaying HA1/L/AcmA recombinant protein, LL-HA1/L/AcmA, to induce mucosal immune responses and to accord protection against influenza virus infection in mice. The LL-HA1/L/AcmA was orally administered into mice and the immune response was evaluated. Mice immunized with LL-HA1/L/AcmA developed detectable specific sIgA in faecal extract, small intestine wash, BAL fluid and nasal fluid. The results obtained demonstrated that oral immunization of mice with LL-HA1/L/AcmA elicited mucosal immunity in both the gastrointestinal tract and the respiratory tract. The protective efficacy of LL-HA1/L/AcmA in immunized mice against a lethal dose challenge with influenza virus was also assessed. Upon challenge, the non-immunized group of mice showed high susceptibility to influenza virus infection. In contrast, 7/8 of mice orally immunized with LL-HA1/L/AcmA survived. In conclusion, oral administration of LL-HA1/L/AcmA in mice induced mucosal immunity and most importantly, provided protection against lethal influenza virus challenge. These results highlight the potential application of L. lactis as a platform for delivery of influenza virus vaccine.

Insertion of single-chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non-recombinant Lactococcus lactis.

Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 ± 1.4% and 3,594.0 ± 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 ± 1.5% and 1,652.0 ± 34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 × 104 molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 × 104 molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154-162, 2017.

Detection of Langat virus by TaqMan real-time one-step qRT-PCR method.

Langat virus (LGTV), one of the members of the tick-borne encephalitis virus (TBEV) complex, was firstly isolated from Ixodes granulatus ticks in Malaysia. However, the prevalence of LGTV in ticks in the region remains unknown. Surveillance for LGTV is therefore important and thus a tool for specific detection of LGTV is needed. In the present study, we developed a real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for rapid detection of LGTV. Our findings showed that the developed qRT-PCR could detect LGTV at a titre as low as 0.1 FFU/ml. The detection limit of the qRT-PCR assay at 95% probability was 0.28 FFU/ml as determined by probit analysis (p ≤ 0.05). Besides, the designed primers and probe did not amplify ORF of the E genes for some closely related and more pathogenic viruses including TBEV, Louping ill virus, Omsk hemorrhagic fever virus (OHFV), Alkhurma virus (ALKV), Kyasanur Forest Disease virus (KFDV) and Powassan virus (POWV) which showed the acceptable specificity of the developed assay. The sensitivity of the developed method also has been confirmed by determining the LGTV in infected tick cell line as well as LGTV- spiked tick tissues.