To explore the effects of the combination of tryptophan (Trp) and arginine (Arg) on growth performance, serum parameters and immune response of broiler chickens challenged with intermediate plus strain of infectious bursal disease virus vaccine, an in vivo experiment was conducted. A corn-soybean meal-based diet containing different levels of Arg and Trp was used. Cobb500 male broiler chickens from 0 to 49 days of age were subjected to a diet supplemented with the combination of Trp and Arg. Growth performance parameters and serum parameters were measured at 27 and 49 days of age. To evaluate the immunomodulatory effects of the combination of Trp and Arg on the challenged chickens, we measured the serum levels of interferon-α, interferon-γ and immunoglobulin G at 27, 35, 42, and 49 days of age. The results showed that the three evaluated immune system parameters including interferon-α, interferon-γ and immunoglobulin G were significantly enhanced after treatment. This enhancement resulted in the recovery of infectious bursal disease virus-infected chickens compared with controls as confirmed by histopathological examinations. Moreover, serum parameters such as albumin and total protein increased, whereas the treatment decreased (P<0.05) the feed:gain ratio, aspartate amino-transferase, alkaline phosphatase, lactic dehydrogenase, triglyceride and cholesterol. These findings suggest that the combination of Arg and Trp has a regulatory effect on growth performance. Moreover, it modulates the systemic immune response against infectious bursal disease.
Previously we have shown that very virulent infectious bursal disease viruses (vvIBDV) that are SspI, TaqI and StyI positive (92/04, 97/61 and 94/B551) but not SspI and TaqI positive and StyI negative (94/273) cause high mortality, up to 80% in specific-pathogen-free chickens with significant damage of the bursal as well as nonbursal tissues. In this study, we sequenced the VP2 gene (1351 bp) of the 92/04, 94/273 and 94/B551 and compared them with other IBDV strains. All the isolates have the unique amino acid residues at positions 222A, 256I, 294I and 299S found in other vvIBDV strains. The deduced VP2 amino acids encoded by 92/04 is identical to the vvIBDV strains from Israel (IBDVKS), Japan (OKYM) and Europe (UK661), whereas the 94/273 and 94/B551 isolates have one to three amino acid substitutions. The 94/273 has two amino acid substitutions at positions 254 G to S and at 270 A to E that have not been reported before from vvIBDV strains. The 94/B551 also has one amino acid substitution at position 300 E to S, which is uncommon among other vvIBDV strains. However, phylogenetic analysis suggested that the isolates are very close to each other and all of them may have derived from the same origin as vvIBDV strains isolated from China, Japan and Europe. Even though antigenic index analysis of the 94/273 and 94/B551 indicated that the isolates are unique compared to other IBDV strains, their antigenic variation remain to be determined by monoclonal antibody study.
The effects of early age feed restriction and heat conditioning on heat shock protein (HSP) 70 expression, antibody production, resistance to infectious bursal disease (IBD), and growth of heat-stressed male broiler chickens were investigated. Chicks were divided into 4 groups: 60% feed restriction on d 4,5, and 6 (FR); exposure to 36 +/- 1 degrees C for 1 h from d 1 to 21 (HT); combination of FR and HT (FRHT); and control. From d 35 to 50, heat stress was induced by exposing birds to 38 +/- 1 degrees C and 80% RH for 2 h/d. On d 36, each bird was administered 10 times the normal dose of live IBD vaccine. After heat exposure, the FRHT birds had higher HSP 70 density (d 41) and weight gain (from d 35 to 49) and lower bursal histological score (BHS) (d 51) than their HT and control counterparts. The HSP 70 expression and BHS of FR birds were not significantly different from those of the other 3 groups during the heat exposure period. Heat shock protein 70 and BHS data were negatively correlated (r = -0.33, P = 0.0008). We concluded that FRHT could improve weight gain and resistance to IBD in male broiler chickens under heat stress conditions. The improved heat tolerance and disease resistance in FRHT birds could be attributed to better HSP 70 response.
Due to the limitations in the range of antibodies recognising avian viruses, quantitative real-time PCR (RT-qPCR) is still the most widely used method to evaluate the expression of immunologically related genes in avian viruses. The objective of this study was to identify suitable reference genes for mRNA expression analysis in chicken intraepithelial lymphocyte natural killer (IEL-NK) cells after infection with very-virulent infectious bursal disease virus (vvIBDV). Fifteen potential reference genes were selected based on the references available. The coefficient of variation percentage (CV%) and average count of these 15 genes were determined by NanoString technology for control and infected samples. The M and V values for shortlisted reference genes (ACTB, GAPDH, HMBS, HPRT1, SDHA, TUBB1 and YWHAZ) were calculated using geNorm and NormFinder. GAPDH, YWHAZ and HMBS were the most stably expressed genes. The expression levels of three innate immune response related target genes, CASP8, IL22 and TLR3, agreed in the NanoString and RNA sequencing (RNA-Seq) results using one or two reference genes for normalisation (not HMBS). In conclusion, GAPDH and YWHAZ could be used as reference genes for the normalisation of chicken IEL-NK cell gene responses to infection with vvIBDV.
Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain.
The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
To gain insights into the role of CD3-/28.4+ intraepithelial lymphocytes-natural killer (CD3-/28.4+IEL-NK) cells during infectious bursal disease virus (IBDV) infection, characterisation of the cells was performed following infection with different strains of the virus. In vitro treatment with IL-18 or ionomycin/PMA successfully stimulated and activated the cells via a significant increase in the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin. Similarly, chickens infected with the vaccine strain of IBDV also up-regulated the expression of CD69, B-Lec, CHIR-AB1 and NK-lysin in CD3-/28.4+ IEL-NK cells up to 3 days post infection (dpi) and down-regulated the expression of the inhibitory receptor B-NK at 3 dpi. On the contrary, infection with the very virulent IBDV (vvIBDV) strain lead to a reduced activation of the cells by down-regulating the expression of the CD69, CHIR-AB1 and NK-lysin especially at 1 dpi. These findings altogether demonstrate the differential activation of CD3-/28.4+IEL-NK cells in chicken following infection with the vaccine or very virulent strains of IBDV. The study therefore provides an important clue into the differential pathogenesis of IBDV infection in chicken. Further studies are however required to determine the functional importance of these findings during IBDV vaccination and infection.