Peste des petits ruminants virus antibodies in domestic large ruminants in Bangladesh.

INTRODUCTION: Peste des petits ruminants (PPR) is an important transboundary animal disease of small ruminants which causes serious damage to the livelihood and food security of millions of small-scale farmers. PPR is endemic in goats in Bangladesh since 1993. The aim of this study was to determine the seroprevalence of PPR in sheep, cattle, and buffaloes in Bangladesh. METHODOLOGY: A total of 434 blood samples from sheep (n = 100), cattle (n = 190) and buffalo (n = 144) were collected aseptically. Sera were separated and antibody titer was determined using a commercially available c-ELISA kit. RESULTS: The overall seroprevalence was 16% and 3.68% in sheep and cattle, respectively, while buffaloes had a considerably higher seroprevalence of 42.36%. The study suggests that buffaloes are more prone to the PPR virus (PPRV) infection and cattle. CONCLUSIONS: This study provides serological evidence of PPRV infection in cattle and buffaloes. These results may warrant further studies to find out the role of large ruminants in transmitting PPRV infection to small ruminants and vice versa and inclusion of all domestic and wild ruminants for regular surveillance program.

Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model.

Francisella tularensis, the causative agent of tularemia, is transmitted by arthropod vectors within mammalian hosts. The detailed mechanisms contributing to growth and survival of Francisella within arthropod remain poorly understood. To identify novel factors supporting growth and survival of Francisella within arthropods, a transposon mutant library of F. tularensis subsp. novicida (F. novicida) was screened using an F. novicida-silkworm infection model. Among 750 transposon mutants screened, the mltA-encoding membrane-bound lytic murein transglycosylase A (MltA) was identified as a novel growth factor of F. novicida in silkworms. Silkworms infection with an mltA deletion mutant (ΔmltA) resulted in a reduction in the number of bacteria and prolonged survival. The ΔmltA strain exhibited limited intracellular growth and cytotoxicity in BmN4 silkworm ovary cells. Moreover, the ΔmltA strain induced higher expression of the antimicrobial peptide in silkworms compared to the wild-type strain. These results suggest that F. novicida MltA contributes to the survival of F. novicida in silkworms via immune suppression-related mechanisms. Intracellular growth of the ΔmltA strain was also reduced in human monocyte THP-1 cells. These results also suggest the contribution of MltA to pathogenicity in humans and utility of the F. novicida-silkworm infection model to explore Francisella infection.

RtxA like protein contributes to infection of Francisella novicida in silkworm and human macrophage THP-1.

Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3-7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells.

Silkworm model for Francisella novicida infection.

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3-7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.

Contribution of methionine sulfoxide reductase B (MsrB) to Francisella tularensis infection in mice.

The essential mechanisms and virulence factors enabling Francisella species to replicate inside host macrophages are not fully understood. Methionine sulfoxide reductase (Msr) is an antioxidant enzyme that converts oxidized methionine into methionine. Francisella tularensis carries msrA and msrB in different parts of its chromosome. In this study, single and double mutants of msrA and msrB were constructed, and the characteristics of these mutants were investigated. The msrB mutant exhibited decreased in vitro growth, exogenous oxidative stress resistance and intracellular growth in macrophages, whereas the msrA mutant displayed little difference with wild-type strain. The double mutant exhibited the same characteristics as the msrB mutant. The bacterial count of the msrB mutant was significantly lower than that of the wild-type strain in the liver and spleen of mice. The bacterial count of the msrA mutant was lower than that of the wild-type strain in the liver, but not in the spleen, of mice. These results suggest that MsrB has an important role in the intracellular replication of F. tularensis in macrophages and infection in mice.