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.

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.

Comparative Pathology of Experimental Avibacterium Paragallinarum Infection in Chicken and Japanese Quail

IntroductionAvibacterium paragallinarum (Av.paragallinarum) is the causative agent of infectious coryza (IC) in chicken, an economically devastating disease of poultry industry. Despite the availability of effective vaccines against IC, the infection is rampant in unorganized poultry sector. Moreover, reports of wider host susceptibility in poultry farms and under field conditions are also emerging. Av.paragallinarum affects the upper respiratory tract of birds and has a predilection for the nasal turbinates. However, no study has systematically evaluated the early pathological changes and mucosal immune responses during Av. paragallinarum infection in the nasal turbinates of chicken. Furthermore, the use of Japanese quail as an alternate experimental animal model than chicken in IC remain unexplored. Here, we examined clinical signs, gross and histopathological changes at the nasal turbinates following experimental Av. paragallinarum infection in chicken and Japanese quail. MethodsWe developed a comprehensive scoring system for recording gross and histopathologic lesions during infection. ResultsOur data show that chicken have a higher susceptibility to Av.paragallinarum infection than the Japanese quail. Japanese quail had significantly lower gross as well as histopathology score in nasal turbinates as compared to infected chicken. Importantly, chicken at an early age i.e. 2.5 weeks were able to induce mucosal immune responses against Av. paragallinarum infection. ConclusionOur findings suggest that there are demonstrable differences in the disease pathology and host immune response to Av. paragallinarum infection in chicken and Japanese quail and warrant further investigation for the use of Japanese quail as an animal model for IC.

Karakteristik Avibacterium paragallinarum Isolat Lapang : Inokulasi pada Telur Ayam Berembrio umur 7 hari

Infectious coryza (IC) adalah penyakit bakterial yang menyerang saluran pernapasan ayam, yang dapat bersifat akut sampai kronis yang disebabkan Avibacterium paragallinarum. IC merusak saluran pernapasan bagian atas, terutama rongga hidung. Pengamatan perubahan makroskopik pada embrio yang mengalami kematian setelah inokulasi pada telur ayam berembrio (TAB) specific pathogenic free (SPF) sebagai salah satu karakteristik isolat A. paragallinarum belum banyak dilakukan. Penelitian ini bertujuan untuk mengetahui karakteristik isolat A. paragallinarum melalui inokulasi pada telur ayam berembrio TAB SPF umur 7 hari. TAB SPF umur 7 hari berjumlah 50 butir dibagi menjadi 5 kelompok masing-masing terdiri dari 10 butir yaitu kelompok kontrol negatif, A. paragallinarum serotipe A/221; serotipe B/Spross; serotipe B/2448; dan serotipe C/2447. Bakteri terlebih dahulu dikultur pada media cair dan diinkubasi selama 24 jam pada suhu 37oC. Suspensi bakteri dengan volume 0,2 ml (6x108 cfu/ml) diinjeksikan pada TAB SPF, diinkubasi pada suhu 37oC dan diamati adanya kematian sebanyak 2 kali sehari. Embrio yang mati dilakukan skoring perubahan makroskopik. Embrio yang berasal dari TAB SPF pada kelompok yang diinokulasi A. paragallinarum serotipe A/ 221, B/Spross, B/2448, and C/2447 mengalami hemoragi dan kekerdilan. Keseluruhan isolat A. paragallinarum bersifat patogen pada telur ayam berembrio.

Macrophage Activation Assays to Evaluate the Immunostimulatory Capacity of Avibacterium paragallinarum in A Multivalent Poultry Vaccine

High-quality vaccines are crucial to prevent infectious disease outbreaks in the poultry industry. In vivo vaccination tests are routinely used to test poultry vaccines for their potency, i.e., their capacity to induce protection against the targeted diseases. A better understanding of how poultry vaccines activate immune cells will facilitate the replacement of in vivo potency tests for in vitro assays. Using the chicken macrophage-like HD11 cell line as a model to evaluate innate immune responses, the current explorative study addresses the immunostimulatory capacity of an inactivated multivalent vaccine for infectious bronchitis, Newcastle disease, egg-drop syndrome, and infectious coryza. The vaccine stimulated HD11 cells to produce nitric oxide and to express pro-inflammatory cytokines IL-1β, TNF, and IL-12p40, chemokines CXCLi1 and CXCLi2, and the anti-inflammatory cytokine IL-10, but only when inactivated Avibacterium paragallinarum, the causative agent of infectious coryza, was present. Lipopolysaccharides from Avibacterium paragallinarum were crucial for the production of nitric oxide and expression of IL-1β and CXCLi1. The described immune parameters demonstrate the capacity of this multivalent vaccine to activate innate immune cells and may in the future, combined with antigen quantification methods, contribute to vaccine quality testing in vitro, hence the replacement of current in vivo vaccination tests.