Campylobacteriosis and Control Strategies against Campylobacters in Poultry Farms.

Campylobacteriosis is a significant foodborne illness caused by Campylobacter bacteria. It is one of the most common bacterial causes of gastroenteritis worldwide, with poultry being a major reservoir and source of infection in humans. In poultry farms, Campylobacters colonize the intestinal tract of chickens and contaminate meat during processing. Vaccines under development against Campylobacters in poultry showed partial or no protection against their cecal colonization. Therefore, this review will elaborate on campylobacteriosis and emphasize the control strategies and recent vaccine trials against Campylobacters in poultry farms. The epidemiology, diagnosis, and treatment of Campylobacter infection, along with specific mention of poultry Campylobacter contamination events in Malaysia, will also be discussed.

Sequence Analysis of the Malaysian Low Pathogenic Avian Influenza Virus Strain H5N2 from Duck.

The avian influenza viruses (AIV) of the H5 subtype have the ability to mutate from low pathogenic (LPAI) to highly pathogenic (HPAI), which can cause high mortality in poultry. Little is known about the pathogenic switching apart from the mutations at the haemagglutinin cleavage site, which significantly contributes to the virus virulence switching phenomenon. Therefore, this study aimed to compare the molecular markers in the haemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of a locally isolated LPAI AIV strain H5N2 from Malaysia with the reference HPAI strains using bioinformatics approaches, emphasising the pathogenic properties of the viral genes. First, the H5N2 strain A/Duck/Malaysia/8443/2004 was propagated in SPF eggs. The viral presence was verified by haemagglutination assay, RT-PCR, and sequencing. Results showed successful amplifications of HA (1695 bp), NA (1410 bp), and M (1019 bp) genes. The genes were sequenced and the deduced amino acid sequences were analysed computationally using MEGA 11 and NetNGlyc software. Analysis of the HA protein showed the absence of the polybasic cleavage motif, but presence of two amino acid residues that are known to affect pathogenicity. There were also two glycosylation sites (glycosites) compared to the reference HPAI viruses, which had three or more at the HA globular head domain. No NA stalk deletion was detected but the haemadsorbing and active centres of the studied NA protein were relatively similar to the reference HPAI H5N2 isolates of duck but not chicken origins. Six NA glycosites were also identified. Finally, we observed a consistent M1 and M2 amino acid sequences between our LPAI isolate with the other HPAI H5N1 or H5N2 reference proteins. These data demonstrate distinct characteristics of the Malaysian LPAI H5N2, compared to HPAI H5N2 or H5N1 from ducks or chickens, potentially aiding the epidemiological research on genetic dynamics of circulating AIV in poultry.

Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

Challenges and Complications of Poly(lactic-co-glycolic acid)-Based Long-Acting Drug Product Development.

Poly(lactic-co-glycolic acid) (PLGA) is one of the preferred polymeric inactive ingredients for long-acting parenteral drug products that are constituted of complex formulations. Despite over 30 years of use, there are still many challenges faced by researchers in formulation-related aspects pertaining to drug loading and release. Until now, PLGA-based complex generic drug products have not been successfully developed. The complexity in developing these generic drug products is not just due to their complex formulation, but also to the manufacturing process of the listed reference drugs that involve PLGA. The composition and product attributes of commercial PLGA formulations vary with the drugs and their intended applications. The lack of standard compendial methods for in vitro release studies hinders generic pharmaceutical companies in their efforts to develop PLGA-based complex generic drug products. In this review, we discuss the challenges faced in developing PLGA-based long-acting injectable/implantable (LAI) drug products; hurdles that are associated with drug loading and release that are dictated by the physicochemical properties of PLGA and product manufacturing processes. Approaches to overcome these challenges and hurdles are highlighted specifically with respect to drug encapsulation and release.

Review of Dendritic Cells, Their Role in Clinical Immunology, and Distribution in Various Animal Species.

Dendritic cells (DCs) are cells derived from the hematopoietic stem cells (HSCs) of the bone marrow and form a widely distributed cellular system throughout the body. They are the most efficient, potent, and professional antigen-presenting cells (APCs) of the immune system, inducing and dispersing a primary immune response by the activation of naïve T-cells, and playing an important role in the induction and maintenance of immune tolerance under homeostatic conditions. Thus, this review has elucidated the general aspects of DCs as well as the current dynamic perspectives and distribution of DCs in humans and in various species of animals that includes mouse, rat, birds, dog, cat, horse, cattle, sheep, pig, and non-human primates. Besides the role that DCs play in immune response, they also play a pathogenic role in many diseases, thus becoming a target in disease prevention and treatment. In addition, its roles in clinical immunology have also been addressed, which include its involvement in transplantation, autoimmune disease, viral infections, cancer, and as a vaccine target. Therefore, based on the current knowledge and understanding of the important roles they play, DCs can be used in the future as a powerful tool for manipulating the immune system.

Regulation of Proteolytic Activity to Improve the Recovery of Macrobrachium rosenbergii Nodavirus Capsid Protein.

The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.

Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response.

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) 'a' determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (ß)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells.

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research.

Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

Virus-like Particle Vaccines: A Prospective Panacea Against an Avian Influenza Panzootic.

Epizootics of highly pathogenic avian influenza (HPAI) have resulted in the deaths of millions of birds leading to huge financial losses to the poultry industry worldwide. The roles of migratory wild birds in the harbouring, mutation, and transmission of avian influenza viruses (AIVs), and the lack of broad-spectrum prophylactic vaccines present imminent threats of a global panzootic. To prevent this, control measures that include effective AIV surveillance programmes, treatment regimens, and universal vaccines are being developed and analysed for their effectiveness. We reviewed the epidemiology of AIVs with regards to past avian influenza (AI) outbreaks in birds. The AIV surveillance programmes in wild and domestic birds, as well as their roles in AI control were also evaluated. We discussed the limitations of the currently used AI vaccines, which necessitated the development of a universal vaccine. We evaluated the current development of AI vaccines based upon virus-like particles (VLPs), particularly those displaying the matrix-2 ectodomain (M2e) peptide. Finally, we highlighted the prospects of these VLP vaccines as universal vaccines with the potential of preventing an AI panzootic.

Negligible effect of chicken cytokine IL-12 integration into recombinant fowlpox viruses expressing avian influenza virus neuraminidase N1 on host cellular immune responses.

In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 105 p.f.u. ml-1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate.

Stealth Coating of Nanoparticles in Drug-Delivery Systems.

Nanoparticles (NPs) have emerged as a powerful drug-delivery tool for cancer therapies to enhance the specificity of drug actions, while reducing the systemic side effects. Nonetheless, NPs interact massively with the surrounding physiological environments including plasma proteins upon administration into the bloodstream. Consequently, they are rapidly cleared from the blood circulation by the mononuclear phagocyte system (MPS) or complement system, resulting in a premature elimination that will cause the drug release at off-target sites. By grafting a stealth coating layer onto the surface of NPs, the blood circulation half-life of nanomaterials can be improved by escaping the recognition and clearance of the immune system. This review focuses on the basic concept underlying the stealth behavior of NPs by polymer coating, whereby the fundamental surface coating characteristics such as molecular weight, surface chain density as well as conformations of polymer chains are of utmost importance for efficient protection of NPs. In addition, the most commonly used stealth polymers such as poly(ethylene glycol) (PEG), poly(2-oxazoline) (POx), and poly(zwitterions) in developing long-circulating NPs for drug delivery are also thoroughly discussed. The biomimetic strategies, including the cell-membrane camouflaging technique and CD47 functionalization for the development of stealth nano-delivery systems, are highlighted in this review as well.

Stingless Bee Honey Improves Spatial Memory in Mice, Probably Associated with Brain-Derived Neurotrophic Factor (BDNF) and Inositol 1,4,5-Triphosphate Receptor Type 1 (Itpr1) Genes.

This study was conducted to evaluate the effects of stingless bee honey (SBH) supplementation on memory and learning in mice. Despite many studies that show the benefits of honey on memory, reports on the nootropic effects of SBH are still lacking, and their underlying mechanism is still unclear. SBH is a honey produced by the bees in the tribe of Meliponini that exist in tropical countries. It features unique storage of honey collected in cerumen pots made of propolis. This SBH may offer a better prospect for therapeutic performance as the previous report identifies the presence of antioxidants that were greater than other honey produced by Apis sp. In this study, SBH was tested on Swiss albino mice following acute (7 days) and semichronic (35 days) supplementation. Experiments were then conducted using Morris water maze (MWM) behaviour analysis, RT-PCR for gene expression of mice striatum, and NMR for metabolomics analysis of the honey. Results indicate spatial working memory and spatial reference memory of mice were significantly improved in the honey-treated group compared with the control group. Improved memory consolidations were also observed in prolonged supplementation. Gene expression analyses of acutely treated mice demonstrated significant upregulation of BDNF and Itpr1 genes that involve in synaptic function. NMR analysis also identified phenylalanine, an essential precursor for tyrosine that plays a role at the BDNF receptor. In conclusion, SBH supplementation for seven days at 2000 mg/kg, which is equivalent to a human dose of 162 mg/kg, showed strong capabilities to improve spatial working memory. And prolonged intake up to 35 days increased spatial reference memory in the mice model. The phenylalanine in SBH may have triggered the upregulation of BDNF genes in honey-treated mice and improved their spatial memory performance.

Shielding of Hepatitis B Virus-Like Nanoparticle with Poly(2-Ethyl-2-Oxazoline).

Virus-like nanoparticles (VLNPs) have been studied extensively as nanocarriers for targeted drug delivery to cancer cells. However, VLNPs have intrinsic drawbacks, in particular, potential antigenicity and immunogenicity, which hamper their clinical applications. Thus, they can be eliminated easily and rapidly by host immune systems, rendering these nanoparticles ineffective for drug delivery. The aim of this study was to reduce the antigenicity of hepatitis B core antigen (HBcAg) VLNPs by shielding them with a hydrophilic polymer, poly(2-ethyl-2-oxazoline) (PEtOx). In the present study, an amine-functionalized PEtOx (PEtOx-NH2) was synthesized using the living cationic ring-opening polymerization (CROP) technique and covalently conjugated to HBcAg VLNPs via carboxyl groups. The PEtOx-conjugated HBcAg (PEtOx-HBcAg) VLNPs were characterized with dynamic light scattering and UV-visible spectroscopy. The colloidal stability study indicated that both HBcAg and PEtOx-HBcAg VLNPs maintained their particle size in Tris-buffered saline (TBS) at human body temperature (37 °C) for at least five days. Enzyme-linked immunosorbent assays (ELISA) demonstrated that the antigenicity of PEtOx-HBcAg VLNPs reduced significantly as compared with unconjugated HBcAg VLNPs. This novel conjugation approach provides a general platform for resolving the antigenicity of VLNPs, enabling them to be developed into a variety of nanovehicles for targeted drug delivery.

Supplementation of Stingless Bee Honey from Heterotrigona itama Improves Antiobesity Parameters in High-Fat Diet Induced Obese Rat Model.

Heterotrigona itama is a common stingless bee species found in Southeast Asia. Studies on the health benefits of its honey are limited in comparison with other stingless bee species. This study examines the antiobesity benefits found in stingless bee honey (SBH) from H. itama. The parameters used to measure the benefits were weight change, morphological structures, and biochemical characteristics. The research was conducted by using rats that were given a high-fat diet (HFD). In total 48 male Sprague Dawley (SD) rats were given a formulated HFD to increase the levels of obesity, the HFD was administered with a value of 0.68 g/cm2. The duration of the treatment was six weeks, and the results show that the induction obesity using the HFD was successful. Following this, the rats were then treated with SBH (at dosages of 1000 mg/kg, 750 mg/kg or 500 mg/kg), with orlistat or with a placebo. Compared with typical obesity treatment methods, the one that used the three dosages of SBH showed a higher reduction in body mass index (BMI), percentage of body weight gain, adiposity index, and relative organ weight (ROW). The levels of liver enzymes (ALT, AST, and ALP) were also significantly lower in SBH-treated groups. The levels of triglycerides and LDL-cholesterol were significantly lower, while the level of HDL-cholesterol was significantly higher in comparison with the control obese group. In terms of morphological structures, the number of adipocyte cells was reduced, and the hepatocytes found in the liver were less prone to rupturing when treated with SBH. In conclusion, the administration of SBH led to an improvement in indicators associated with obesity reduction. SBH also possesses a hepatoprotective potential which can reduce the health risks related to obesity.