Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

A multiplex (m)PCR and a PCR followed by restriction fragment length polymorphism (RFLP) analysis of Avibacterium paragallinarum have been proposed as alternatives to conventional serotyping by the Page scheme. We evaluated both methods, and also sequenced the PCR-RFLP target fragment to reexamine the capacity of molecular serotyping. Eleven reference strains and 27 field isolates were used. Many reference strains and isolates were misidentified as Page serogroup B. The sequence analysis revealed 6 profiles based on the matching rates of the target sequence with the 3 reverse primers of the mPCR. The reference strains and field isolates in profiles 1 and 4 were correctly identified as serogroup A or C by the mPCR. The strains and/or isolates in profiles 2, 3, 5, and 6 could be misidentified as serogroup B or as nontypeable by the mPCR. The homology comparison of the sequences showed that the target sequence of the mPCR, called region 2, was not Page serogroup specific, although some Kume serovars, such as A-1 and C-2, were correctly serotyped. In addition, there was a 9 nucleotide deletion in the sequences of profiles 1, 3, and 5, but not of profiles 2, 4, and 6. Overall, we confirmed that the mPCR and PCR-RFLP molecular assays are not suitable for identifying the serogroups of A. paragallinarum isolates. With further study, analysis of region 2 sequences may have potential as a means of recognizing the Kume serovars of A. paragallinarum isolates.

Avibacterium paragallinarum is the pathogen of infectious coryza, which is a highly contagious respiratory disease of chickens that brings a potentially serious threat to poultry husbandry. Iron is an important nutrient for bacteria and can be obtained from surroundings such as siderophores and hemophores. To date, the mechanisms of iron acquisition and heme utilization as well as detailed regulation in A. paragallinarum have been poorly understood. In this study, we investigated the transcriptomic profiles in detail and the changes of transcriptomes induced by iron restriction in A. paragallinarum using RNA-seq. Compared with the iron-sufficiency control group, many more differentially expressed genes (DEGs) and cellular functions as well as signaling pathways were verified in the iron-restriction group. Among these DEGs, the majority of genes showed decreased expression and some were found to be uniquely present in the iron-restriction group. With an in-depth study of bioinformatic analyses, we demonstrated the crucial roles of the Hut protein and DUF domain-containing proteins, which were preferentially activated in bacteria following iron restriction and contributed to the iron acquisition and heme utilization. Consequently, RT-qPCR results further verified the iron-related DEGs and were consistent with the RNA-seq data. In addition, several novel sRNAs were present in A. paragallinarum and had potential regulatory roles in iron homeostasis, especially in the regulation of Fic protein to ensure stable expression. This is the first report of the molecular mechanism of iron acquisition and heme utilization in A. paragallinarum from the perspective of transcriptomic profiles. The study will contribute to a better understanding of the transcriptomic response of A. paragallinarum to iron starvation and also provide novel insight into the development of new antigens for potential vaccines against infectious coryza by focusing on these iron-related genes.

Avibacterium paragallinarum is the pathogen that causes infectious coryza, a highly contagious respiratory disease that brings a serious threat to chickens. Heme utilization systems play an important role in bacterial adversity adaptation and pathogenicity, and our previous report found the presence of heme utilization (HutZ) in Av. paragallinarum . However, little is known about the function of HutZ in Av. paragallinarum . In this study, the HutZ mutant strain of Av. paragallinarum was successfully developed and identified by PCR and western blot analysis. Mutation of HutZ significantly retards bacterial growth under reduced iron conditions, indicating the regulatory role of HutZ on growth and iron acquisition. Notably, the HutZ mutant strain had slower growth than the wild-type strain when heme was provided as the sole source of iron; thus, HutZ is crucial for heme utilization in Av. paragallinarum . Moreover, the HutZ mutant strain exhibited a markedly compromised tolerance to acid stress compared to the wild-type strain. Pathogenicity analysis showed that mutation of HutZ significantly weakened the ability of bacteria to invade and reproduce in host macrophage cells in vitro . Furthermore, the HutZ mutation could significantly decrease the bacterial virulence in chickens, which displayed lower morbidity and milder clinical symptoms. Hence, this is the first study to demonstrate in-depth the essential roles of HutZ on iron homeostasis and pathogenesis of Av. paragallinarum , which provides novel insight into advances of new prophylactic vaccines against this kind of bacteria. Importance: Heme utilization (HutZ) protein has been characterized as an important heme-degrading enzyme that is critical for the cleavage of heme to biliverdin via verdoheme and can release iron to be used by bacteria. The interaction between HutZ and Av. paragallinarum is still unknown. Here, we unraveled the role of HutZ on the growth, iron acquisition, heme utilization, and resistance to acidic stress in Av. paragallinarum . We also uncovered the importance of HutZ for the success of Av. paragallinarum infection and provided new clues to the pathogenesis strategies of this organism. This work constitutes a relevant step toward an understanding of the role of HutZ protein as a master virulence factor. Therefore, this study is of great importance for understanding the mechanisms underlying Av. paragallinarum virulence and may contribute to therapeutic applications.