Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens

Abstract

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

Intracloacal Inoculation of Broiler Chickens with Clostridium perfringens Strains: Evaluation of Necrotic Enteritis Disease Development and Lymphoid Immune Responses

“…To this end, the present study found that the broiler birds infected intracloacally with virulent CP18 and CP26 strains showed a significantly increased CT expression of IL-1β, IL-6, and IFNγ compared to the uninfected control and avirulent CP5-infected groups. This observation was in support of our previous studies that host inflammatory responses are often mounted against the virulent, but not avirulent, strains [ 4 , 18 ]. However, this response in CT appeared to be highly regulated when birds were infected intracloacally, as demonstrated by an increased expression of anti-inflammatory/ immunoregulatory cytokines or downregulation of expression of pro-inflammatory cytokines in the mucosal lymphoid tissues, including CT, bursa, and HG.…”

“…For example, broiler chickens coinfected with E. maxima and virulent C. perfringens induced an increased intestinal gene expression of pro-inflammatory cytokines (IL-1β, IFNγ, IL-17, IL-8, and others) [ 16 , 36 ], while in vitro stimulation of chicken intestinal epithelial cells with virulent C. perfringens or purified alpha-toxin led to an increased transcription of IL-6, IL-8, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF), and nuclear factor kappa-B (NFkB) genes [ 37 ]. In support of these findings, macrophages have been shown as the likely source of such mucosal inflammatory responses [ 18 , 38 ] [Kulkarni, et. al., 2023; unpublished].…”

“…Subsequent melt curve analysis was performed by heating to 95 °C for 15 sec, cooling to 60 °C for 1 min, and heating to 95 °C for 15 sec. Primers for the amplification of TLR21, IL-1β, IL-4, IFNγ, IL-6, IL-10, IL-13, and TGFβ [ 18 , 21 ] were synthesized by Integrated DNA Technologies (Coralville, IA, USA), and the primer sequences along with the gene-specific annealing temperature are given in Table 1 . Using the formula to quantify the relative amount of gene expression that is E of the target standard curve (Cp of target calibrator) − (Cp of target samples) / E of reference standard curve (Cp of reference samples) − (Cp of reference calibrator) , the expression levels of target genes were calculated with the housekeeping gene, β-actin reference gene, as described previously [ 22 , 23 ].…”

“…This observation indicated that immune responses against C. perfringens were spatially regulated and that the inflammatory response depended on the level of pathogen virulence. In another study, we also found that macrophages may be the cellular source of inflammatory cytokines induced in response to virulent C. perfringens [ 18 ]. In the present study, we asked if intracloacal inoculation of broiler chickens with the same C. perfringens strains (CP5, CP18, and CP26) and the dietary predisposition model used previously [ 4 ] would impact the severity of NE development and its associated mucosal and systemic lymphoid immune responses.…”

Effect of CpG-Oligonucleotide in Enhancing Recombinant Herpes Virus of Turkey-Laryngotracheitis Vaccine-Induced Immune Responses in One-Day-Old Broiler Chickens

“…Single-cell splenocyte preparations (n = 8 per group) were made following a protocol described previously [ 25 ]. Briefly, the single cells were plated on 96-well round-bottom plates with each well containing 10 6 cells in 100 μL FACS buffer (PBS with 1% BSA).…”