In this study, the authors examined the technical performance of culture methodology using specific media: Mycoplasma isolation media of pleuropneumonia-like organisms (PPLO) broth and PPLO agar. Digitonin sensitivity, growth inhibition, the serum plate agglutination test, a commercially available enzyme-linked immunosorbent assay (ELISA) and a commercially available simplex polymerase chain reaction (PCR) test were used to detect Mycoplasma gallisepticum infections in samples collected from the lungs, trachea and tracheal swabs of poultry. These samples were collected from broiler-breeder flocks, broiler flocks and layer flocks. In addition, genomic bacterial deoxyribonucleic acid (DNA) was extracted and amplified, using a simplex PCR. The seroprevalence of M. gallisepticum antibodies in chickens and chicks was also investigated. The prevalence of M. gallisepticum was found to be highest in the layer flocks, at 33.3% (17/51), when the tracheal swab procedure was adopted. In young birds, the serum plate agglutination test and ELISA assay detected antibodies against M. gallisepticum in 69.9% (320/458) and 58.3% (267/458) of the chicken samples, respectively, and 48.7% (146/300) and 60% (180/300) of the samples from the chicks.
The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.
Colon cancer remains one of the main cancers causing death in men and women worldwide as certain colon cancer subtypes are resistant to conventional treatments and the development of new cancer therapies remains elusive. Alternative modalities such as the use of viral-based therapeutic cancer vaccine is still limited, with only the herpes simplex virus (HSV) expressing granulocyte-macrophage colony- stimulating factor (GM-CSF) or talimogene laherparepvec (T-Vec) being approved in the USA and Europe so far. Therefore, it is imperative to continue the search for a new treatment modality. This current study evaluates a combinatorial therapy between the oncolytic Newcastle disease virus (NDV) and interleukin-12 (IL-12) cytokine as a potential therapeutic vaccine to the current anti-cancer drugs. Several in vitro analyses such as MTT assay, Annexin V/FITC flow cytometry, and cell cycle assay were performed to evaluate the cytotoxicity effect of recombinant NDV, rAF-IL12. Meanwhile, serum cytokine, serum biochemical, histopathology of organs and TUNEL assay were carried out to assess the anti-tumoral effects of rAF-IL12 in HT29 tumor-challenged nude mice. The apoptosis mechanism underlying the effect of rAF-IL12 treatment was also investigated using NanoString Gene expression analysis. The recombinant NDV, rAF-IL12 replicated in HT29 colon cancer cells as did its parental virus, AF2240-i. The rAF-IL12 treatment had slightly better cytotoxicity effects towards HT29 cancer cells when compared to the AF2240-i as revealed by the MTT, Annexin V FITC and cell cycle assay. Meanwhile, the 28-day treatment with rAF-IL12 had significantly (p < 0.05) perturbed the growth and progression of HT29 tumor in NCr-Foxn1nu nude mice when compared to the untreated and parental wild-type NDV strain AF2240-i. The rAF-IL12 also modulated the immune system in nude mice by significantly (p < 0.05) increased the level of IL-2, IL-12, and IFN-γ cytokines. Treatment with rAF-IL12 had also significantly (p < 0.05) increased the expression level of apoptosis-related genes such as Fas, caspase-8, BID, BAX, Smad3 and granzyme B in vitro and in vivo. Besides, rAF-IL12 intra-tumoral delivery was considered safe and was not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney. Therefore, this study proves that rAF-IL12 had better cytotoxicity effects than its parental AF2240-i and could potentially be an ideal treatment for colon cancer in the near future.
Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB) sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE) range (DE 10–14) of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p < 0.01) than maltodextrin with higher DE ranges (DE 15–19 and DE 20–24) and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application.
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