ABSTRACT
Background and Aim: Fowl adenovirus (FAdV) 8b causes inclusion body hepatitis, resulting in major economic losses globally among chickens. The objectives were to inactivate FAdV 8b isolate propagated in chicken embryo liver (CEL) cells using a stirred tank bioreactor (UPM08136P5B1) and determine the humoral and cell-mediated immune response, efficacy, and virus shedding in broiler chickens. Materials and Methods: The FAdV 8b isolate UPM08136P5B1 was inactivated using binary ethyleneimine, adjuvanted with Montanide 71VG, inoculated into day-old broiler chickens in a booster group (BG) and non-booster group (NBG), and challenged with a pathogenic FAdV 8b strain. Clinical signs, gross lesions, body weight (BW), liver: body weight ratio, FAdV antibody titer using enzyme-linked immunosorbent assay, and histopathological changes were recorded. The CD3+, CD4+, and CD8+ T-lymphocyte profiles of the liver, spleen, and thymus using flow cytometry, and viral load in liver and cloacal shedding using quantitative polymerase chain reaction were evaluated. Results: Chickens in the challenged control group (CCG) exhibited mild clinical signs, gross lesions, and histopathological changes, which were absent in the inoculated groups, and had lower BW and higher liver BW ratio than chickens in the unchallenged control group (UCG); BG and NBG on 35- and 42-days post-inoculation (DPI). Chickens in NBG and BG had higher antibodies than UCG on 7, 21, 35, and 42 DPI. The challenged BG and NBG produced higher antibodies than the CCG on 35 DPI. T-lymphocytes were higher among the inoculated groups than UCG in the liver, spleen, and thymus. Inoculated challenged groups recorded higher CD3+, CD4+, and CD8+ T-lymphocytes on 35 and 42 DPI than CCG. The challenged control group had a significantly higher viral load in the liver than challenged that in BG on 35 DPI and BG and NBG on 42 DPI. The challenged control group had significantly higher challenge FAdV shedding than challenged inoculated groups on 35 and NBG on 42 DPI. Conclusion: UPM08136P5B1 was successfully inactivated and mixed with Montanide 71VG. The inactivated vaccine candidate that induced humoral and cellular immunity was effective, reduced FAdV load in the liver, and shedding in the cloaca, and could be useful against FAdV 8b infections in chickens.
ABSTRACT
Aims@#Psittacine birds such as parrots, macaws, cockatoos, lovebirds and parakeets, are widely reared as household pets or at aviary due to their attractive features. However, the status of virus-causing diseases of psittacine species in Malaysia is fairly under-documented. Therefore, this study was aimed to detect the presence of three common avian viruses that infect psittacine birds, i.e. beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus. @*Methodology and results@#Faecal samples from twelve asymptomatic captive psittacine birds of different species were collected from an undisclosed animal garden in Serdang, Selangor, Malaysia. Briefly, the sample was homogenised and resuspended with SM buffer with the ratio 1:1 (weight of sample/g: volume of SM buffer/mL) before centrifugation at 1,000 × g for 20 min. The supernatant was collected and filtered before subjected to genomic DNA extraction using a commercialised kit. Polymerase chain reaction (PCR) technique was used to screen the V1, VP1 and L1 genes of beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus, respectively. Findings revealed that the samples were negative for BFDV and avian polyomavirus. However, positive results of 1.5 kbp PCR amplicon were detected for avian papillomavirus in four out of the 12 samples (33.33%), which was from the white-crested cockatoo, African grey parrot, yellow-collared macaw and Senegal parrot. Sequence analysis of the L1 gene from the Senegal parrot Poicephalus senegalus revealed 93% identity to a reference Psittacus erithacus timneh avian papillomavirus.@*Conclusion, significance and impact of study@#This study added to the limited prevalence data of three important avian viruses which infect captive psittacines in Seri Kembangan, Selangor, Malaysia. Avian papillomavirus, but not BFDV and avian polyomavirus, was detected in the collected captive psittacine birds. Therefore, a routine screening can be performed to monitor the health status of birds despite their asymptomatic manifestation, in order to prevent possible virus transmission.
Subject(s)
Virus Diseases , BirdsABSTRACT
@#Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was constructed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (ΔUPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.
