Construction of an Attenuated Pasteurella Multocida B:2 by Mutation in the Gdha Gene

Pasteurella multocida 8:2 is a Gram negative bacteria that has been associated with haemorrhagic septicaemia in cattle and buffaloes in Asia. It has been known to produce endotoxin that leads to haemorrhages and oedema, causing deaths due to either asphyxiation and dyspnoea or septicaemia. Vaccination has been used to control the disease but with little success due to the low vaccination coverage. Therefore, an alternative live vaccine should be considered. In preparing an alternative live vaccine, an attenuated P. multocida 8:2 is created by manipulating one of the housekeeping genes of the bacteria. The selected housekeeping gene, the glutamate dehyrogenase (gdhA) gene, was successfully isolated via PCR from wild type P. multocida 8:2. The gene was then amplified using nested-PCR to determine its functional part. Both PCR products were cloned into plasmid pCR2.1, producing pSZ1 and pSZ2, respectively before being sequenced. The whole sequence of the gene is 1108 bp while the functional part of the gene was 652 bp. The functional part was 99.8% identical to the model sequence, the PM70, which is a model genome sequence of P. multocida serotype A. The pSZ 1 was subsequently digested with a unique restriction enzyme, Munl before the kanamycin cassette, isolated from plasmid pUC4K via PCR, was inserted at the centre of the housekeeping gene. The recombinant was named pSZ1K. After that, the gdhA gene that was disrupted by kanamycin cassette (GK) was isolated from the pSZ1K using restriction enzyme digestion, EcoRI. The suicide plasmid, pAKA19 was also digested with the same enzyme to achieve complimentary ligation sites. After ligation, the achieved recombinant plasmid was called pSZ19GK. All cloning products were transformed into Escherichia coli DH5a. Enroute for disruption of the gene in the host genome, both E. coli and P. multocida 8:2 were subjected to spontaneous mutation towards streptomycin. After conveying the pSZ19GK into P. multocida 8:2 via conjugation, the bacteria was incubated for five days to encourage allelic exchange to occur between disrupted gene and the host chromoso me. Subsequently, PCR of the bacteria genome proved that allelic exchange has occurred and the mutant was called P. multocida 8:2 (GK). In order to verify the characteristic of the non-pathogenic P. multocida 8:2 (GK) mutant, in vitro stability test and in vivo pathogenicity test were done. In in vitro stability test, 14 strains out of the 20 survived only up to 15 days of incubation. This proves that the mutants are unable to sustain life without glutamate supplement and therefore having a short life-span. From there, several strains were picked to be tested in vivo using mouse experimental model. Mice infected intraperitoneally or subcutaneously with different concentrations of the mutant survived throughout the 5-day study period. They were compared to the mice that were infected intraperitoneally or subcutaneously with different concentrations of the wild type organism. None of the mice infected with the mutant died but all mice infected with the wild type did not survived and were dead in less than 24 hours. P. multocida 8:2 were successfully isolated from organs of mice infected with both wild-type and mutant. This confirmed that the mutant, P. multocida 8:2 (GK) became attenuated by the disruption of the gdhA gene and has a good potential to be used as an alternative live vaccine for HS

Interactions of an attenuated AroA- derivative of Pasteurella multocida B:2 with mammalian cells and its potential for DNA vaccine delivery

The primary aim of this study was to investigate the potential of an aroA mutant of Pasteurella multocida B:2 (vaccine strain JRMT12) as a candidate for DNA vaccine delivery (bactofection). First, the invasive property of the vaccine strain was assessed for its interaction with different mammalian cell lines. Next, a eukaryotic expression plasmid that could be maintained in Pasteurella was modified to contain a prokaryotic reporter gene to help in determining the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of the mammalian cells. This plasmid was further developed to function with a dual prokaryotic and eukaryotic reporter system in order to demonstrate expression of the plasmid DNA in the mammalian cells. During interaction of strain JRMT12 with mammalian cell lines, the ability of the bacterium to adhere, invade and survive intracellularly was monitored and assessed. Three mammalian cell lines were used: a mouse macrophage-like cell line, J774.2; a bovine-lymphoma cell line, BL-3; and an embryonic bovine lung cell line, EBL. The JRMT12 strain was compared with strains of the wild-type P. multocida B:2 (85020), bovine P. multocida A:3, Mannheimia haemolytica A:1 and Escherichia coli XL-1 BLUE. Both P. multocida B:2 strains were capable of adhering to and invading J774.2, BL-3 and EBL cells. All of the Pasteurella and Mannheimia strains tested were able to adhere to EBL cells but only B:2 strains were taken up intracellularly in significant numbers. The vaccine strain, JRMT12 was found to survive intracellularly in EBL cells for at least 7 h although a steady decline in the number of viable intracellular bacteria was noted with time. In an invasion inhibition assay, the use of the microfilament formation inhibitor cytochalasin D suggested that the entry into mammalian cells was by an actin-dependent process. Cell viability assessment by trypan blue staining indicated that none of the bacterial strains was toxic for the mammalian cells. Upon entry into the mammalian cells the JRMT12 strain resided in a vacuolar compartment, as demonstrated by transmission electron microscopy. However, P. multocida A:3 and M. haemolytica A:1 were only found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. Further morphological assessment by TEM showed that only a low percentage of mammalian cells appeared to contain one or more JRMT12, suggesting that only certain cells in the population were capable of being invaded by, or taking up, the bacteria. Attempts were made to construct a Pasteurella eukaryotic expression plasmid using a gene sequence from the Pasteurella shuttle plasmid pAKA16, developed previously in this laboratory, and the commercial eukaryotic expression plasmid pCMV-sCRIPT, but these were only partially successful. The origin of replication gene (oriP) in the Pasteurella shuttle plasmid was isolated and sequenced. Analysis of oriP showed sequence similarity with the known origins of replication in other Pasteurella plasmids. The E. coli plasmid origin of replication (oriE) was removed from pCMV-sCRIPT and the oriP gene was ligated into the oriE-free pCMV-sCRIPT but attempts to transform the resulting plasmid into P. multocida B:2 were not successful. An alternative approach to plasmid development was made using another commercial eukaryotic expression vector, pEGFP-N1. This plasmid has the same properties as pCMV-sCRIPT but has an additional, fluorescent reporter gene under the control of a eukaryotic promoter. It was found to be able to replicate in P. multocida B:2 but positive transformants were only recovered after prolonged incubation after electroporation. The plasmid was stably maintained in strain JRMT12 for at least 14 days with or without antibiotic selection. It was also successfully transfected into EBL cells, as shown by expression of green fluorescent protein (GFP) in individual cells. The P. multocida vaccine strain JRMT12 was also able to deliver the plasmid into EBL cells, although the number of EBL cells expressing GFP after bacterial delivery was lower than by direct transfection of the plasmid. Next, plasmid pMK-Express, a Pasteurellaceae prokaryotic expression vector with a gfp reporter gene, was used. When this was electroporated into the vaccine strain, the strain was shown to express GFP maximally as measured by fluorimetry, during the early exponential phase of bacterial growth. The DsRed.M1 gene coding for red fluorescent protein (RFP) from plasmid pDsRed-Monomer was then used to replace the gfp gene in pMK-Express to make the construct pMK-RED. After electroporation of pMK-RED into the JRMT12, RFP expression was detected maximally during the early exponential phase of bacterial growth. The same strain expressing RFP could also be detected in the intracellular compartment of the EBL cells by fluorescence microscopy at 3 h post-invasion. Finally, plasmid pSRG, our so-called “traffic light” plasmid with a dual reporter system was constructed. This was made from plasmid pEGFP-N1 (with its existing eukaryotic expression system for GFP expression) and the sodRED fragment (with a Pasteurella promoter controlling the DsRED.M1 gene for RFP expression) isolated from plasmid pMK-RED. This plasmid was stable in strain JRMT12 with or without antibiotic selection for 14 passages. RFP expression from JRMT12 was detected maximally during the early exponential phase of bacterial growth. Transfection of pSRG into EBL cells gave individual cells expressing GFP. Invasion assays with EBL cells and P. multocida B:2 JRMT12 pSRG+ showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously. At 5 h post-invasion, some of the EBL cells were still harbouring RFP-expressing bacteria and at the same time expressing GFP themselves. Concurrently, some Pasteurella free-EBL cells were shown to express GFP. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine. An apparent immunosuppressive effect of P. multocida B:2 on the proliferative response to concanavalin A (ConA) of peripheral blood mononuclear cells (PBMC) had been reported by Ataei (2007). The PBMC had been taken from calves infected with P. multocida B:2 or from normal calves and treated in vitro with extracts of P. multocida B:2. In the present study, in vitro assays with PBMC from normal calves were undertaken in an attempt to confirm these findings. A cell-free extract (CFE) of the vaccine strain JRMT12 was found to suppress the subsequent proliferation of PBMC in response to ConA in a dose-dependent manner. However, the results were not consistently reproducible and the same effect could not be demonstrated with CFE from the wild-type strain 85020

Comparison of analgesic efficacy between preemptive intravenous paracetamol and single shot caudal block in paediatric inguinal hernia repair

Terdapat pelbagai kaedah untuk mencapai tahap keberkesanan analgesik yang mencukupi selepas pembedahan angin pasang di kalangan kanak-kanak. Pembiusan setempat ‘caudal block’ biasanya digemari dalam pembedahan bahagian bawah abdomen melibatkan kanak-kanak. Sebaliknya, paracetamol yang diberi melalui vena telah digunakan secara meluas kerana tahap keselamatan dan keberkesanan analgesiknya yang sangat baik. Kajian ini bertujuan untuk membandingkan tahap keberkesanan analgesic paracetamol yang diberi melalui vena dengan pembiusan setempat ‘caudal block’ selepas pembedahan angina pasang di kalangan kanak-kanak. Ini adalah satu kajian prospektif rawak melibatkan 40 orang kanak-kanak yang menjalani pembedahan elektif sebelah angin pasang di bawah pembiusan am di Hospital Universiti Sains Malaysia. Pesakit yang telah mendapat keizinan dari penjaga mereka dibahagi secara rawak kepada dua kumpulam yang menerima paracetamol melalui vena (kumpulan A) dan pembiusan setempat ‘caudal block’ (kumpulan B). Skor kesakitan diukur menggunakan skala FLACC pada 10 minit, 30 minit, 1 jam, 2 jam dan 6 jam selepas pembedahan dan dibandingkan. Keputusan lain yang diukur and dibandingkan adalah purata tekanan darah dan denyutan nadi semasa pembedahan, purata masa pertama untuk permintaan ubat tahan sakit, purata jumlah fentaniyl yang digunakan semasa tempoh pembedahan dan kadar kesan sampingan antara kedua-dua kumpulan Purata umur pesakit adalah 30.48 bulan. Pesakit dalam kumpulan B mendapat keputusan skor kesakitan yang lebih rendah pada 10 minit (p=0.018), 30 minit (p=0.013) dan 1 jam (p=0.05) dibandingkan dengan kumpulan A. Pada 2 jam dan 6 jam, tiada perbezaan yang signifikan direkodkan. Tiada ketidakstabilan hemodinamik direkodkan. Tiada perbezaan signifikan dalam masa pertama untuk permintaan ubat tahan sakit (p=0.079) dan juga jumlah total penggunaan fentanyl (p=0.090) semasa tempoh pembedahan. Tiada komplikasi besar yang signifikan diperhatikan pada kedua-dua kumpulan. Pembiusan setempat ‘caudal block’ merupakan pilihan yang lebih baik daripada paracetamol yang diberi melalui vena pada jam pertama selepas pembedahan namun tidak memberi perubahan yang signifikan selepas tempoh tersebut. Keberkesanan analgesik paracetamol yang diberi melaui vena adalah sama dengan pembiusan setempat ‘caudal block’ jika dikira dari perspektif masa pertama permintaan ubat tahan sakit dan juga jumlah penggunaan fentanyl

Managing Flood in the Context of Education, Research and Partnership in Malaysia

Several structural and non-structural measures and approaches have been implemented by the Malaysian government in order to overcome and alleviate the flood disaster. To date, Education, Partnership and Research have been identified as crucial components in forming an effective Flood Management system in Malaysia. This argument has been supported by many public and private institutions worldwide. As such, the standards and plans can be developed and implemented at district, state and federal levels. The spirits of Hyogo and Sendai were used as the guiding principles. Semi-structured interviews with several responding agencies were conducted for data collection. The findings indicated that less emphasis was given to the roles of components such as Education, Partnership and Research in Flood Management. The aim of this paper is to propose an integrated system for storing, disseminating and analyzing information pertaining to Education, Partnership and Research

A study on unsafe act, unsafe condition and communication barrier for fall from height accident in construction industry

Malaysian construction industry growth is expected to fluctuate since it was announced by the Finance Minister as part of the infrastructure project for Malaysian Budget 2020. In Malaysia, construction industry is one of the sectors which contribute to fall from height accidents. It is one of the important issues that need to be addressed by every organization in order to understand the impacts on the organizations including the workers. Currently, fall from height accidents is a challenge that many organizations struggle to overcome. Thus, to gain better understanding, this study identifies the factors that influence fall from height accidents. The main purpose of this study is to investigate the safety problem when working at height in construction site, to identify the factors that cause fall from height accidents and to analyze the factors that influence the accident and the prevention. For data collection, 264 set of questionnaires were distributed to 12 different construction sites with two (2) target levels which is supervisors and site workers. The data collected were analyze using SPSS Version 26 and the result gain through analysis of Pearson correlation and Multiple regression indicated that unsafe act, unsafe condition and communication barrier have positive relationship with fall from height accident. These finding provides useful information to the organization regarding their employees’ well-being. Besides that, it helps to enhance the underpinning theory in this study which is Accident Causation Model

Investigating tight junction proteinrelated regulation of nasal epithelial barrier integrity in allergic rhinitis patients and non-allergic individuals

Allergic rhinitis (AR) is an allergic disease affecting a huge population worldwide. Despite not being fatal, it has a significant impact on the quality of life. The nasal epithelial barrier is considered crucial for the first line of defence in the upper airway as it protects the host immune system from exposure to allergens. Defective tight junctions (TJs) contribute to nasal epithelial barrier dysfunction in moderate-severe AR patients. However, the association of nasal epithelial TJs, zonula occludens (ZO) proteins and histone deacetylases (HDACs) with the demographical, clinical and environmental characteristics of AR patients remains unclear. In this study, we aimed to investigate the mRNA expression of ZO-1, ZO-2, ZO-3, HDAC1 and HDAC2 in nasal epithelial cells of house dust mites (HDMs)-sensitised AR patients compared to non-allergic controls. We also examine the association between ZO-1, ZO-2, ZO-3, HDAC1 and HDAC2 levels in nasal epithelial cells with the demographical, clinical and environmental parameters of AR patients and non-allergic controls. The recruited subjects consisted of 28 AR patients and 28 non-allergic controls. A Skin prick test (SPT) was performed on the subjects to determine whether they were allergically sensitised to either one of the HDM allergens. We started this study by collecting nasal epithelial cell samples from all the subjects. The RNA samples were reverse transcribed into cDNAs for measurement of ZO-1, ZO-2, ZO-3, HDAC1 and HDAC2 expression levels by quantitative real-time polymerase chain reaction (qRT-PCR). The mRNA expression of ZO-1 was significantly decreased in AR patients compared to non-allergic controls (p=0.010). No significant difference was observed in the expression levels of ZO-2, ZO-3, HDAC1 and HDAC2 in AR patients compared to non-allergic controls. However, we found a significant association of higher HDAC2 levels in AR patients sensitised to Dermatophagoides farinae (D. farinae) (p=0.041). We also found significant associations of higher HDAC2 levels in AR patients with lower frequency of changing bedsheet (p=0.043). Higher expression of ZO-2 was observed in AR patients who had pets (p=0.007). In conclusion, our data indicated that ZO-1 expression was lower in AR patients, contributing to decreased nasal epithelial barrier integrity. In addition, we also demonstrated a correlation between the mRNA expression of ZO-2 and HDAC2 in nasal epithelial cells with specific environmental parameters. Furthermore, the presence of allergens in the bedsheet also leads to an increase in HDAC2 expression, which may affect ZOs expression in nasal epithelial barrier. Targeting the nasal epithelial barrier by restoring ZO-1 expression may be a promising therapeutic approach for AR patients

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs

V protein, the virulence factor across the family Paramyxoviridae: a review

Paramyxoviridae is a family of viruses within the order Mononegavirales and comprises 14 genera; Metaavulavirus, Orthoavulavirus, Paraavulavirus, Synodonvirus, Ferlavirus, Aquaparamyxovirus, Henipavirus, Morbillivirus, Respirovirus, Jeilongvirus, Narmovirus, Salemvirus, Pararubulavirus and Orthorubulavirus. The members within this family are negative and single-stranded RNA viruses including human and animal pathogens such as measles virus (MeV), Nipah virus (NiV), mumps virus (MuV), Sendai virus (SeV) and Newcastle disease virus (NDV). The V protein is conserved within the family and plays an essential role in viral pathogenicity. Although V proteins of many paramyxoviruses are interferon-antagonists which counteract with the host’s innate immunity, there are still differences in the mode of action of the V protein between different genera or species within the same genera. The strategies to circumvent the host interferon (IFN) pathway can be divided into three general mechanisms; degradation of signal transducers and activators of transcription (STAT) protein, inhibition of phosphorylation of the transcription factor and, inhibition of translocation of STAT proteins into the nucleus. As a result, inhibition of IFN signalling and production promotes viral replication in the host cells. This review highlights the mechanism of the paramyxoviral V protein in evading the host IFN system

Horizontal Gene Transfer: A Vehicle for the Dissemination of Resistance and Virulence Determinants during Colonization and Disease

The successful in vivo horizontal transfer of mobile genetic elements carrying resistance and virulence determinants have contributed immensely to a global dissemination of virulent and multi-drug resistant pathogens. In addition, the pathogenesis of MRSA infection is enhanced via initial colonization of the skin through the component of the microbial surface antigen recognizing adhesive matrix molecules and by their ability to evade host immune response. Furthermore, it was also observed that the genetic diversity of pathogenic MRSA is due to its’ ability to rapidly acquire resistance and virulence determinants. A characteristic feature that made it one of the most important nosocomial pathogen worldwide. Similarly, the expression of virulence gene in MRSA has been observed to be regulated by the accessory gene regulator system (agr). These system is made up of a series of genes whose product build up quorum-sensing regulatory mechanisms that is growth dependent. In addition, at a certain growth stage, the agr systems triggers a pronounced changes in the expression of genes called the quorum sensing. The findings of this review affirms the importance of horizontal gene transfer in the dissemination of resistance and virulence determinants and as well as the genetic diversity of MRSA

Lactococcus lactis: LAB model organism for bacteria-mediated therapeutic strategies

Lactococcus lactis is a well-characterized, food-grade lactic acid bacterium (LAB) with generally recognized as safe (GRAS) status. Better understanding of this bacterium at a molecular level has led to the development of unprecedented genetic tools that enable the expression of heterologous proteins. Subsequently, the ability of L. lactis to express and deliver these proteins to eukaryotic hosts presents a promising approach to achieve potent treatments for various diseases. Here, we have reviewed the characteristics of L. lactis and the expression systems established for this LAB model organism. We also described the experimental applications of L. lactis in disease therapy, especially its role as a vector in vaccination strategies