Novel NAD‐independent Avibacterium paragallinarum: Isolation, characterization and molecular identification in Iran

Abstract Background Infectious coryza (IC) is an invasive upper respiratory disease caused by Avibacterium paragallinarum that affects birds, particularly chickens. The objective of this study is to isolate, characterize and molecularly identify the bacterium A. paragallinarum in poultry birds, as well as to determine its antibiotic sensitivity and resistance. Methods A total of 10 chickens from four different Iranian farms with typical IC symptoms were used in this study. The nasal swabs were streaked onto chocolate agar plates and blood agar plates and incubated at 37°C in 5% CO2 for 24 to 48 h. As part of the identification of bacteria, bacteriological observations and polymerase chain reaction (PCR) testing are conducted. The antibiotic sensitivity tests were also performed using the disk diffusion method against A. paragallinarum and the prevalence in different farms was determined. Results By using biochemical assays and PCR analyses, seven strains of A. paragallinarum were isolated from samples of four chicken farms with typical IC clinical signs. Most isolates (4/7) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. Three of the seven strains of A. paragallinarum were found to be novel NAD‐independent under anaerobic conditions. There was one biochemical biovar identified in terms of carbohydrate fermentation patterns, although changes in maltose carbohydrate fermentation patterns were detected in the No. 5 strain. All isolates were sensitive to gentamicin and spectinomycin. Three novel NAD‐independent strains (Nos.1, 5 and 7) were found to be multidrug‐resistant (MDR) and resistant to at least three classes of antibiotics. There was greater antibiotic resistance in the three NAD‐independent isolates than in normal NAD‐dependent bacteria. Conclusion By discovering NAD‐independent forms of A. paragallinarum, these species have a greater range than previously believed. A clear, cautious approach should be taken in diagnosing and possibly controlling IC.


INTRODUCTION
Infectious coryza (IC), a disease of the upper respiratory system in chickens, is caused by Avibacterium paragallinarum (previously Haemophilus paragallinarum) (Ali et al., 2013). The incidence of IC is increasing in many parts of the world, affecting chickens of all ages, both indigenous (native chickens) and laying hens in poultry farms (Paudel et al., 2017). In addition, this disease affects developing hens and layers and is economically significant since it increases culling rates and decreases egg output (by between 10 and -40%). (Ali et al., 2013;Crispo et al., 2018;Paudel et al., 2017). In the acute phase of the disease, facial swelling, inflammation of the sinuses and conjunctiva and purulent discharge from the nose characterize the disease (Crispo et al., 2018). Beach et al. thought of IC as a separate clinical entity as early as 1920. The common etiological agent was unknown for many years because the disease was frequently misdiagnosed in mixed infections, particularly with chicken pox (Blackall et al., 1997). Bacillus hemoglobinophilus coryzae gallinarum was named after the causal agent by DeBlieck (1932) (Blackall et al., 1997). The binomial Haemophilus gallinarum was proposed by both Elliot andLewis andDelaplane et al. in 1934 (Schalm &Beach, 1936). Due to its demand for both x-(haemin) and v-(nicotinamide adenine dinucleotide, NAD) factors for development, the causal agent of IC was identified as H. gallinarum during research done in the 1930s (Feberwee et al., 2019).
IC is a major economic challenge in many regions of the world. The infection is most prevalent in California and the South-eastern states of the United States (Blackall et al., 1997). IC is probably available all around the world. Acute catarrhal inflammation of the nasal passages and sinuses is caused by A. paragallinarum. Catarrhal conjunctivitis and subcutaneous oedema of the face and wattles are common occurrences. Pneumonia and airsacculitis are uncommon in broilers; nevertheless, certain reports of prevalence in broilers have revealed high levels of contamination (up to 69.8%) due to airsacculitis, even without other viral or bacterial infections (Muhammad & Sreedevi, 2015).
At present, there is no definitive treatment available for this condition, and antibiotics can only reduce the severity of the symptoms, and they cannot remove bacteria, so once the antibiotic is stopped, the disease will recur (Jeong et al., 2017).
The use of vaccinations helps to prevent and control infectious diseases such as coryza. In spite of immunization, there is still a risk of acquiring the disease (Deshmukh et al., 2015). It is possible to identify and isolate the causal agent based on symptoms, post-mortem findings and bacteriological tests of the causative agent, thereby enabling diagnosis (Vargas & Terzolo, 2004). Most isolates of A. paragallinarum require NAD as a growth factor (Badouei et al., 2014). NADindependent isolates and isolates with unusual growth properties have been identified as well. The inability to ferment D-xylose and trehalose, as well as the lack of catalase, differentiates A. paragallinarum from the other Avibacterium species (Badouei et al., 2014). The diagnosis is made based on clinical symptoms, and the confirmation is made by biochemical testing. A rapid, accurate and precise diagnosis using polymerase chain reaction (PCR) is fundamental for avoiding coryza damage (Nouri et al., 2020). The prevalence of the disease has increased from 14 to 25% in recent years, prompting global preventative efforts (Han et al., 2016). Coryza instances increased significantly in California in 2017, according to the California Animal Health and Food Safety Laboratory System, and adequate herd vaccination and biosafety standards should always be established and updated based on these findings. However, there is no specific information on the prevalence of this disease in Iran (Banani et al., 2007). The Razi Institute (Nouri et al., 2020) provided the latest information on the disease diagnosis in 2020. Meanwhile, clinical symptoms of suspected IC are common in Iranian commercial poultry, and treatment with various medicines is done without completing an antibiogram and imported IC vaccination is widely used. However, research on IC and its agent is rare in Iran, with just a few reports of IC agent isolation and identification (Ahmadi & Nasri, 2018;Banani et al., 2007;Nouri et al., 2020). The present study aimed to isolate, molecularly identify and investigate the biochemical properties and antibiotic susceptibility of A. paragallinarum bacteria. The collected samples were cultured in the laboratory for less than 2 h to avoid any bacterial inactivation.

Antimicrobial susceptibility testing
The antimicrobial sensitivity test was carried out utilizing the Chuki-

Case history
As shown in Table 2, efforts to isolate A. paragallinarum were performed by swabbing the infraorbital sinus of birds from poultry farms with a history of reduced egg production.
Swelling of the infraorbital sinus, oedema of the surrounding tissues, occasionally spreading to the wattles and hyperaemia of the conjunctiva are all common findings in the instances that were presented. Eyelid and wattle oedema ranged from severe to minor (Figure 1).

Bacterial isolation and biochemical identification
Information about the appearance of bacteria and bacterial colonies on solid culture media (chocolate agar plates) is given in this field (Figure 2). The appearance of clear, dew-like colonies on chocolate agar indicated the field strain of A. paragallinarum. Co-culture of strains sus-pected of A. paragallinarum with S. epidermidis ATCC 14990 also showed larger colonies close to the Staphylococcus line. This indicates haemolysis and release of factor V from red blood cells by Staphylococcus, which results in increased growth of A. paragallinarum and is an initial confirmation of A. paragallinarum isolation.
Seven field isolates were obtained and identified as A. paragallinarum using Gram staining, and biochemical testing between 2020 and 2021 ( Table 3). All of the isolates were Gram-negative, catalase-deficient and could not grow on MacConkey agar. D-mannitol, maltose generated acid in all strains, but not D-xylose or trehalose (Table 3). Variable acid generation from maltose and D-xylose was observed, allowing three biochemically unique biovars to be identified based on carbohydrate fermentation patterns. However, differences in maltose carbohydrate fermentation patterns were discovered in the No. 5 strain. The majority of the isolates (4 of 7) needed NAD and 5% CO 2 , but three of them (Nos.1, 5 and 7) were NAD-independent and developed on blood agar in anaerobic conditions.

Molecular identification of A. paragallinarum
Primers were designed for this study using Primer Express bioinformatics software. The forward and reverse primers of Table 2    paragallinarum were identified and used for antimicrobial sensitivity.

Antimicrobial susceptibility
An antibiogram test was used to evaluate antimicrobial susceptibility. According to the antibiotic susceptibility Table 4, all seven A. para-gallinarum strains were susceptible to gentamicin and spectinomycin (100%). The results of this test showed that one, five and seven bacterial strains are resistant to more than three antibiotic classes, so that these bacteria are a subset of multi-drug resistant (MDR) bacteria.
The pattern of antibiotic resistance of the tested strains is recorded in Table 4.

Prevalence assessment
In A. paragallinarum species-specific PCR assays, 35 out of 40 (87.5%) of examined birds were found to be infected with the A. paragallinarum.

DISCUSSION
IC has a nearly worldwide distribution, and to our knowledge, there is little information on the disease's precise frequency among industrial breeder flocks and/or backyard poultry in Iran (Badouei et al., 2014).
The persistence of the IC agent among chicken farms is thought to be due to multi-age poultry manufacturing systems, partial immunity provided by serovar content in commercial vaccines against regionally prevalent A. paragallinarum serovars, and the role of backyard chicken as a source of IC infection for nearby chicken flocks (Gallardo et al., 2020). In spite of occasional reports of IC from Iran, no molecular surveillance data are available to describe the epidemiology of the disease in our surrounding nations (Badouei et al., 2014). Officially, no other studies (Ahmadi & Nasri, 2018;Badouei et al., 2014;Banani et al., 2007;Blackall & Soriano-Vargas, 2020;Blackall et al., 1990;Falconi-Agapito et al., 2015;Gallardo et al., 2020;Han et al., 2016;Luna-Galaz et al., 2016;Nouri et al., 2020;Vargas & Terzolo, 2004), the result of the biochemical identification method was the isolation of Gram-negative, catalase-negative, oxidase-negative coccobacilli, which were d-xylose and trehalose-negative in fermentation, but were able to ferment Dmannitol and maltose. These are some of the biochemical features of bacteria that cause IC (Blackall & Soriano-Vargas, 2020). However, differences in maltose carbohydrate fermentation patterns were discovered in the No. 5 strain. The majority of the isolates (4 of 7) needed NAD and 5% CO 2 , but three of them (Nos.1, 5 and 7) were NAD-independent and developed on blood agar in anaerobic conditions. This is the only research that we are aware of that reports the identification of various A. paragallinarum growth variations in Iran. NAD-independent A.
paragallinarum isolates have been found in South Africa, Mexico and Peru thus far (Falconi-Agapito et al., 2015;Garcia et al., 2004). The two NAD-independent isolates in this investigation were from two separate commercial layer farms and one from a broiler breeder farm, with three of them coming from probable IC cases that had been treated with antibiotics. A. paragallinarum was verified by PCR using 16S ribosomal RNA-specific primers. PCR assays can be used to simplify the diagnostic process after initial isolation and biochemical identification (Badouei et al., 2014). Another benefit of this procedure is its quickness since the findings are available in less than 24 h. The PCR method was utilized to diagnose coryza in chickens in this study. All seven of the suspicious isolates acquired from culture produced the A. paragallinarum unique band (165 bp). The efficacy of accurate identification of IC purely based on culture method from a choanal cleft in naturally infected chicken is reflected in the biochemical method's result when compared to PCR detection. This finding is similar to Chen et al. (1996), who found that PCR testing in natural conditions returned 15 of 39 birds and 6 of 8 commercial farms positive, compared to 8 of 39 birds and 4 of 8 commercial farms positive via culture (Chen et al., 1996).  (Chukiatsiri et al., 2012;Priya et al., 2012). Previous research suggests that NAD-independent isolates may be able to escape host immunological responses, which might result in vaccination failure if the vaccine is based on a NAD-dependent A. paragallinarum strain (Bragg et al., 1997;Jeong et al., 2017). However, the relevance of NAD-independent isolates in vaccination failures is unclear, as a commercial trivalent vaccine based on typical NAD-dependent strains has been found to protect against challenge by NAD-independent isolates under experimental conditions (Jacobs & Van der Werf, 2000). Diagnostic laboratories must also be aware of the possibility of A. paragallinarum strains that are not dependent on NAD. Ornithobacterium rhinotracheale, another catalase-negative, Gram-negative bacterium, might readily be mistaken for these NAD-independent isolates (Jeong et al., 2017 (Han et al., 2016;Siddique et al., 2012). In contrast, A. paragallinarum was found to be present in 87.5% of samples suspected of being infected with IC in the current investigation, which indicates the high prevalence of A. paragallinarum.

CONCLUSIONS
Using biochemical assays and PCR analyses, seven strains of A. paragallinarum were isolated from samples of four chicken farms with typical IC clinical signs. Three of the seven strains of A. paragallinarum were found to be NAD-independent and MDR. In this study, the isolation, identification and antibiotic pattern of A. paragallinarum are critical in providing options for coryza disease control. Reports from field cases indicate that commercial vaccines have not been able to cover the prevalence of IC in Iran. While screening for antibiotic susceptibility, provides a beneficial suggestion for an appropriate therapy that is successful and efficient against bacterial infection. Molecular characteristics of A. paragallinarum obtained using PCR aid in the rapid diagnosis of these bacteria. The frequency of A. paragallinarum in broiler and layer hens in Iran was found to be 87.5% in the current investigation.
To rule out the existence of A. paragallinarum in chickens in Iran, more study with bigger sample sizes and better sampling methods is needed in different seasons and geographical locations.