Isolation, Serovar Identification, and Antimicrobial Susceptibility of Avibacterium paragallinarum from Chickens in China from 2019 to 2020

Infectious coryza is an acute infectious respiratory disease in chickens that is caused by Avibacterium paragallinarum (A. paragallinarum). Infectious coryza has major economic effects due to decreased egg production in growing birds and slowed growth in broilers. In this study, we isolated and identified 40 strains of A. paragallinarum from chickens that showed typical clinical signs of coryza in part of China from 2019 to 2020. Using a hemagglutination-inhibition test, 11 isolates were identified as serovar A, 10 isolates were identified as serovar B, and 19 isolates were identified as serovar C. Antimicrobial sensitivity tests showed that high minimum inhibitory concentration (MIC) values were encountered for compounds sulfamethoxine sodium and oxytetracycline hydrochloride. Especially, of the 40 A. paragallinarum isolates, 30% had an MIC value of compound sulfamethoxine sodium of 64 μg/mL, 10% of 128 μg/mL, and 15% of 256 μg/mL. For oxytetracycline hydrochloride, 85% of isolates showed MIC values of 64 μg/mL or more. Excitingly, the MIC values of β-lactamase (amoxicillin, ampicillin, and ceftiofur) were low, with 77.5%, 70%, and 92.5% of isolates having an MIC value of ≤1 μg/mL, respectively. Our results may provide a reference for the treatment of infectious coryza.

Short Communication: PCR detection of Avibacterium paragallinarum from layers with infectious coryza symptoms in poultry farms of Sleman District, Indonesia

Fauziah I, Asmara W, Wahyuni AETH, Widayanti R. 2021. Short Communication: PCR detection of Avibacterium paragallinarum from layers with infectious coryza symptoms in poultry farms of Sleman District, Indonesia. Biodiversitas 22: 4890-4894. Infectious coryza (IC), caused by Avibacterium paragallinarum, is a contagious and infectious respiratory tract disease that affects the commercial poultry industry. Molecular techniques, such as species-specific PCR, HPG-2 PCR are mostly used for the detection of A. paragallinarum. The current research was carried out to isolate A. paragallinarum from the layers of infectious coryza signs in Sleman District, special region of Yogyakarta, followed by PCR confirmation of the identified bacteria. Nine field isolates were observed and determined based on their colony and cell morphology. All isolates were characterized biochemically and confirmed with species-specific HPG-2 PCR for A. paragallinarum. Out of 9 isolates, 6 (66.7%) isolates were biochemically identified as A. paragallinarum and confirmed by HPG-2 PCR.

Reports of Infectious Coryza in Peafowls (Pavo cristatus) at Solapur District of Maharashtra, India

Background: Infectious coryza is a bacterial disease caused by Avibacterium paragallinarum which causes acute respiratory disease in chickens but it is also reported in Indian peafowls. Although this is a treatable condition in peafowls but need to diagnose and treat as earliest possible to save peafowls. The aim is to present reported cases of infectious coryza in peafowls. Methods: From 2017 to 2020 total seven cases of peafowls showing symptoms of swollen eyes, sticky discharge from eyes and nostrils, closed eye lids due to swelling, purulent discharge from eyes and caseous masses were also observed while pressing of swelling over eyes were reported from different part of Solapur district. Gasping was common symptom observed in all the peafowls. All the cases were confirmed as infectious coryza on the basis of symptoms, though the ocular and nasal swabs were submitted for bacterial isolation to the laboratory. All peafowls were treated by using parenteral antibiotic injection Gentamicin @ 4.4 mg/kg of body weight and non-steroidal anti inflammatory drugs (NSAID) injection Meloxicam @ 0.2 mg/kg of body weight and cleaning of eyes with distilled water and instillation of gentamicin and diclofenac eye drops, oral administration of multivitamins and energy drinks Result: Out of seven cases, 6 were female and 1 was male peafowl. Recovery rate was 85.71% and all recovered peafowl were released back in the nature. Recovery was depending on severity of symptoms and all six birds recovered within one to three weeks’ time.

A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.

Antimicrobial sensitivity of Avibacterium paragallinarum isolates from layers in the special region of Yogyakarta, Indonesia

Background and Aim: Infectious coryza (IC) is an upper respiratory disease of chicken caused by Avibacterium paragallinarum. Its clinical symptoms are swollen face and malodorous sinus exudate. This study was conducted to determine the antimicrobial sensitivity of A. paragallinarum isolates from layers in the Special Region of Yogyakarta, Indonesia. Materials and Methods: The samples used in this study were 30 layers that showed IC symptoms. The colony and cell morphology were observed with Gram staining; then, biochemical tests (catalase, oxidase, urease, indole, and motility tests, and carbohydrate fermentation tests using lactose, maltose, mannitol, and sorbitol) were performed to the suspected colony to identify A. paragallinarum. An antibiotic sensitivity test was performed using several antibiotic disks against A. paragallinarum isolates that were cultured on Mueller-Hinton Agar. Results: Out of 30 samples, 24 samples (80%) were found positive for A. paragallinarum. All isolates were sensitive to ampicillin (AMP) and amoxicillin (AML) (100%), and chloramphenicol (C) (91.6%). The antibiotics with intermediate sensitivity were enrofloxacin (79.2%), fosfomycin (75%), and ciprofloxacin (54.2%). The isolates were most resistant to erythromycin (100%), followed by tetracycline (87.5%), streptomycin (83.3%), doxycycline and kanamycin (70.8%), and trimethoprim (62.5%). Conclusion: Out of the total samples, 24 samples (80%) from layers with IC symptoms were identified biochemically as A. paragallinarum. It was sensitive to AMP, AML, and C.

A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

Resident bacteria contribute to opportunistic infections of the respiratory tract

Opportunistic pathogens frequently cause volatile infections in hosts with compromised immune systems or a disrupted normal microbiota. The commensalism of diverse microorganisms contributes to colonization resistance, which prevents the expansion of opportunistic pathogens. Following microbiota disruption, pathogens promptly adapt to altered niches and obtain growth advantages. Nevertheless, whether and how resident bacteria modulate the growth dynamics of invasive pathogens and the eventual outcome of such infections are still unclear. Here, we utilized birds as a model animal and observed a resident bacterium exacerbating the invasion of Avibacterium paragallinarum (previously Haemophilus paragallinarum) in the respiratory tract. We first found that negligibly abundant Staphylococcus chromogenes, rather than Staphylococcus aureus, played a dominant role in Av. paragallinarum-associated infectious coryza in poultry based on epidemic investigations and in vitro analyses. Furthermore, we determined that S. chromogenes not only directly provides the necessary nutrition factor nicotinamide adenine dinucleotide (NAD+) but also accelerates its biosynthesis and release from host cells to promote the survival and growth of Av. paragallinarum. Last, we successfully intervened in Av. paragallinarum-associated infections in animal models using antibiotics that specifically target S. chromogenes. Our findings show that opportunistic pathogens can hijack commensal bacteria to initiate infection and expansion and suggest a new paradigm to ameliorate opportunistic infections by modulating the dynamics of resident bacteria.

The Transcriptomic and Bioinformatic Characterizations of Iron Acquisition and Heme Utilization in Avibacterium paragallinarum in Response to Iron-Starvation

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.