Initial Characterization of the FlgE Hook High Molecular Weight Complex of

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility

Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo

Activation of Human Monocytes by Live Borrelia burgdorferi Generates TLR2-Dependent and -Independent Responses Which Include Induction of IFN-β

It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-β and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-α, IL-6, IL-10 and IL-1β in monocytes than did lysates. Secreted IL-18, which, like IL-1β, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-β and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs

Reproductive Parameters of Native Sows in Selected Area of Bangladesh

Reproductive performance is considered as one of the important factors affecting the productivity and economy of the pig farms. The objective of the study was to determine the reproductive parameters of gilts and sows by direct interview method using a pretested questionnaire in selected area of Bangladesh. A total of 20 pig farms having 60 gilts and 27 sows were included in this investigation. The present study revealed that, in native female pigs, the age at puberty was 229.0 ± 16.7 days, the oestrous cycle length was 22.2 ± 1.2 days, the oestrous duration was 37.9 ± 14.2 hours, the gestation length was 113.5 ± 1.0 days, the interval between farrowing and onset of oestrus was 62.9 ± 11.9 days and the number of piglets born per native sow was 5.25 ± 2.3. It was also found that the first service pregnancy rate was 71.7% and the number of services required for each pregnancy was 1.3. The highest proportion of pigs was detected in oestrus by observing stand to be mounted by boars. It is concluded that reproductive parameters of native sows need to be improved for better production.DOI: http://dx.doi.org/10.3329/bjvm.v12i2.21294 Bangl. J. Vet. Med. (2014). 12 (2): 211-215  </jats:p

Detection of 19-bp indel of PLAG1 gene and its effects on morphometric traits in indigenous and crossbred cattle of Bangladesh

Stature and live weight are economically important traits in livestock species and polygenic in nature. Pleomorphic adenoma gene 1 (PLAG1) is a member of the PLAG family of zinc finger transcription factors which has a specific association with growth related traits in human and various livestock species including cattle. This study investigated the 19-bp indel (insertion/deletion) polymorphism of the PLAG1 gene and its possible association with morphometric traits in indigenous and Holstein-Local (H×L) crossbred cattle of Bangladesh. A total of 160 blood samples were collected from the aforementioned cattle populations whereas data on morphometric traits were recorded only from H×L crossbreds. PCR amplification fragment harboring 19-bp indel detected three genotypes II (366 bp), ID (366/347 bp) and DD (347 bp). The II, ID and DD genotype frequencies were 0.00, 0.09 and 0.91 respectively in indigenous cattle populations and the corresponding frequencies were 0.14, 0.43 and 0.43, respectively in H×L crossbred populations. None of the investigated morphometric traits had a significant association with 19-bp indel genotypes. However, non-genetic factors such as herd, year and parity had significant effects only on hip height trait in crossbred cattle (P&lt;0.001). In conclusion, the identified 19-bp indel polymorphism was temperate breed Holstein specific and was absent in indicine populations that could be utilized only for crossbred cattle of Bangladesh upon validation with large a number of samples.&#x0D; Bangladesh Journal of Animal Science. 51(1):31-39. &#x0D;  </jats:p