Isolation, Identification and Antimicrobial Susceptibility of Avibacterium Paragallinarum from Backyard Chicken in Retail Markets of Karaj and Tehran, Iran

1. Introduction

Avibacterium (Haemophilus) paragallinarum (Av. Paragallinarum) is the causative agent of infectious coryza (IC), an important disease of chicken associated with an acute upper respiratory infection, growth retardation, and reduced egg production ( 1 ). IC is normally characterized by an acute onset and spreads rapidly in a flock with the symptoms of nasal discharge, facial swelling, lacrimation, anorexia, and diarrhea ( 1 , 2 ).

Unusual clinical symptoms of IC are similar to those of swollen head syndrome reported among chickens in the United States ( 2 , 3 ). In principle, the prevention of IC mostly relies on sanitation practices, secluded sites, and vaccination in poultry farms. Despite these, the sporadic outbreak of infectious coryza is still widespread which causes significant economic losses, especially in developing countries. Despite the worldwide distribution of IC and familiarity of the farmers with the disease, no systematic study, to the best of our knowledge, was conducted on isolation and characterization of Av. Paragallinarum in Iran. Bozorgmehri fard ( 4 ) conducted the first study of antibiotic sensitivity of the pathogenic bacteria. Banani, Pourbakhsh ( 5 ) studied isolation and characterization of the pathogenesis of Av. Paragallinarum from infected layer flocks. In general, IC often occurs in backyard chickens in Iran. Therefore, a study should be conducted on isolation and identification of Av. Paragallinarum to manage and prevent IC among these chickens in the future.

2. Material and Methods

2.1. Samples Collection

A total of 18 choanal swabs samples were obtained from backyard chicken suspected of IC infection for bacterial isolation and detectionand transferred to Brain Heart Infusion (BHI) broth media supplemented with Nicotinamide Adenine Dinucleotide (NAD) (.0025mg/ml). The birds with facial swelling, nasal discharge, and respiratory disorder were selected for sampling from retail markets of Karaj and Tehran between November 2018 to May 2019. The obtained samples were cultured in a laboratory for less than 2 hoursto prevent possible bacterial deactivation.

2.2. Bacterial Isolation

The samples were cultured witha swab streaked on 5% sheep blood agar supplemented with 1% heat-inactivated, sterile-filtered chicken serum and then followed by cross-streaking of Staphylococcus aureus as NAD provider for media. Bacterial growth was supported by incubation at37° C and 5% CO₂ pressure for 24-48 hours. Bacterial colonies whichindicated satellite growth were selected to subculture in 7% horse blood Columbian chocolate agar for further purification and maintenanceas recommended ( 6 ). Finally, gram staining negative colonies were biochemically screened for catalase-negative and oxidase-positive tests.

2.3. Detection and Identification of Av. Paragallinarum by PCR

DNAs required for the test were extracted from both cultured bacterial colonies and swab samples. The DNA extractions from bacterial culture were performed following the methods as described by Moore ( 7 ) via boiling an appropriate amount of clone purified isolates in sterile distilled water for 45-60 minutes. The swab samples were prepared for DNA extraction after wrapping the wets wabs in media around the tube vessel, the remained transport media were centrifuged at 13,000 rpm and the obtained precipitate was harvested in 400µl of sterile distal water. The DNA extraction was followed by the phenol-chloroform method as previously described ( 7 , 8 ). In performed experiments, a previously characterized Av. Paragallinarum and Ornithobacte riumrhinotracheale were includedas positive and negative bacterial genome control, respectively.

The extracted DNAs from both swab and bacterial samples were used in the test of HPG-2 PCR specific for Av. Paragallinarum, as described by Chen, Miflin ( 9 ). The primers, N1 and R1 (Table 1) set to generate an amplicon of approximately 500bp in the final electrophoretic analysis. Furthermore, the middle third (hypervariable region) of the hemagglutinin gene sequence of isolates were amplified and sequenced for deeper identification and verification of isolated bacteriausing published primer sets of 5-1 forward and 5-1 reverse with the size of approximately 1.6 Kbp. All PCR tests were performedin 25-ul volumes containing an appropriate amount of template DNA (15-150 ng), MgCl₂, and primer concentrations using Eppendorf® Mastercycler® thermal cycler. PCR products were visualized by UV light after electrophoresis in 1% agarose gel for 25 min at 80V.

2.4. Antibacterial Susceptibility Test

Av. Paragallinarum is a slow-growing and fastidious bacterium. Most of its strains require V-(nicotinamide adenine dinucleotide, NAD) factor for their growth and there is no recommended standardized medium for susceptibility testing. Therefore, Columbia blood agar (CBA) with 7% horse blood was used to perform antibacterial susceptibility test by Kirby-Bauerdisk diffusion method following the guidelines of Clinical and Laboratory Standard Institute. A suspension of the fresh culture of isolates (of approximately 1 × 10⁸ CFU/mL) was prepared in BHI broth supplemented with NAD which was adjusted to 0/5 McFarland turbidity standard, then evenly spread onto CBA in a Petri dish. Then, 20 commonly used antibiotics in farms were selected for investigating antibiotic susceptibility. All antimicrobial discs were from antibiotic discs of Patan-Teb, Iran which were aseptically placed onto the surface of inoculated agar plate media. Zones of growth inhibition around each of the antibiotic discs were measured to the nearest millimeter after incubation (16–24 h at 37 °C with 5% CO₂ pressure). Using the reference table of the company, the size of the zone of growth inhibition related to each antibiotic was recorded as: "the isolate is susceptible (S), intermediately susceptible (I), or resistant (R)".  Antibiotics and their potencies were as follow: ampicillin (10 μg), amoxicillin (25 μg), colestin (10μg), cephalexin (30μg), ceftriaxone (30 μg), doxycycline (30μg), danofloxacin (5 μg), erythromycin (15 μg), florfenicol (30 μg), flumequine (30 μg) fosfomycin (200 μg), gentamycin (10 μg), lincospectin (20μg), neomycin (30 μg), enrofloxacin (5 μg), ofloxacin (5 μg), penicillin (10 μg), streptomycin (10 μg), trimethoprim-sulfamethoxazol (1.25 + 23.15 μg), oxytetracycline (30 μg), tiamulin (30 μg) and tylosin (30 μg).

3. Results

Av. Paragallinarum bacteria were isolated from 4 samples of 18 choanal swab samples by showing typical morphological and biochemical characteristics of satellite growth, Grams negative polymorphic short rod bacteria, oxidase activity, and non-catalase reaction. Cultivation of positive samples generally appeared in form of tiny dewdrops and non-hemolytic colonies even in the first round of cultivations (Figure 1). Meanwhile, these isolates had also no growth in culture media of MacConkey agar and at normal air incubation.

Figure 1. Satellite growth of Avibacterium paragallinarum around strike culture of Staphylococcus aureus on sheep blood agar plate.

Molecular detection and identification of these isolates were confirmed by observing the expected 500bp band in electrophoresis of their species-specific PCR products and sequencing partial hemagglutinin protein gene. After proper alignment of obtained partial nucleotide sequences of related hemagglutinin gene of isolates, they were deposited at the Genbank genetic sequence database (http://www.ncbi.nlm.nih.gov/genbank) by accession number (MN928965-8). In Molecular tests by Av. Paragallinarum species-specific PCR, 12of 18 (66%) of sampled birds were infected with Av. Paragallinarum.

Also, antimicrobial susceptibility tests revealed that all four Av. Paragallinarum isolates were resistant to amoxicillin, oxytetracycline, streptomycin, trimethoprim/ sulfamethoxazole (up to 75%) and sensitive to cefalexin, ceftriaxone, enrofloxacin, florfenicol, gentamycin, linco-spectin, neomycin, doxycycline (50%), danofloxacin (75%), flumequine (50%), ofloxacin (75%). An intermediate growth inhibition zone was observed around antibiotic discs for ampicillin, colistin, erythromycin, penicillin, tiamulin (75%), tylosin (75%) according to the reference table of the company (Patan-Teb, Iran).

4. Discussion

Infectious Coryza is almost universally distributed and to the best of our knowledge, no information exists on the exact prevalence of the disease among commercial breeding herds and/or backyard chicken in Iran. It is believed that the persistence of IC agents in poultry farms could be based on the multi-age poultry production system and partial immunity due to serovar content of commercial vaccine against regional prevalent serovars of Av. Paragallinarum and also the role of backyard chicken as a source of IC infection for nearby chicken flocks ( 1 , 2 ).

Despite some reports of IC from Pakistan, no molecular monitoring data exists to describe the epidemiology of IC in the neighboring countries of Iran. The occurrence of IC in commercial poultry flocks of Iran has not been officially reported by the Iran Veterinary Organization, which could beat some point a good outcome of vaccination protocols performed by the imported vaccines against serogroups A, B, and C during the last decades. However, in recent years, the clinical picture of IC has been unofficially reported in some layer farms as IC was bacteriologically confirmed by isolation of causative bacteria by authors in one case (not published yet). Generally, IC appears to be controlled in poultry farms due to the vaccination used in most sensitive poultry flocks particularly, layer and breeder chicken. However, the disease in chicken of rural regions and live bird marketing venues frequently has been a challenging clinical issue for veterinarians. Av. Paragallinarum has been isolated from different organs of diseased birds such as the infraorbital sinus, choanal cleft, trachea, nasal secretion, and internal organs of the liver, air sacs ( 1 , 10 ). In this study, chickens presented in the retail live market were selected with typical clinical symptoms of conjunctivitis, unilateral or bilateral facial swelling, and infra-orbital sinus swelling and nasal secretion. Sample birds were notably around 6-8 months old that had been separately taken to market from different parts of the country in groups of 20 to 50 birds. The results of bacterial culture from 18 groups of swab samples obtained from each suspected group of birds resulted in the isolation of four Av. Paragallinarum (22 %). All isolates were V-factor dependent which presented typical ‘satellite’ growth on solid plate cultures. The result of the culture method (22%) compared to PCR detection (66%) reflects the low efficiency of accurate detection of IC only based on the culture method of the choanal cleft in naturally infected chicken. This result is comparable to the report of Chen et al. (1996) as the PCR test in the normal condition was positive in15/39 birds and 6/8 commercial farms while the positive result of culture was observed in 8/39 birds and 4/8 farms. Another experimental challenge study concluded that Av. Paragallinarum could be detected in 50% or less of chickens as positive at 21 and 24 days post-challenge in sinus swab samples by both PCR and culture method and PCR detection and culture methods indicated similar results up to 18 days post-challenge ( 11 ). The sensitivity of the PCR method in detecting Av. Paragallinarum appears to depend on the time of sampling and nature of the infection. Recently, Clothier, to rain ( 12 ) reported the frequency of Av. Paragallinarum infection using real-time PCR in 294 samples from small poultry flocks submitted to the California Animal Health and Food Safety Laboratory System (Davis, CA) among which 86 (30%) samples were PCR-positive for Av. Paragallinarum. Therefore, it seems that molecular genomic detection in field conditions by PCR or culture methods is less sensitive than in the case of experimental conditions. Selection of the main respiratory organs (infra-orbital sinus, trachea) of birds for Av. Paragallinarum isolation is critical. Meanwhile, considerable attention should be paid to avoid the deletion in sampling times in respect to the onset of the disease in birds and rolling out the other disease conditions with similar clinical picture to IC to reduce the discrepancies in efficiency of diagnostic methods.

In the present study, as expected, according to the microbiota of the buccal cavity of birds, along with Av. Paragallinarum isolation, other bacteria such as hemolytic Gram-positive (staphylococcus. spp) and Gram-negative colonies of coliform-like bacteria has also been identified beside Ornithobacterium rhinotracheale. Apart from reported avian viral pathogens that may contribute to the pathogenesis of IC ( 1 , 2 , 12 ), mycoplasma gallisepticum ( 1 , 13 ), Ornithobacterium rhinotracheale ( 14 ), Gallibacteriumanatis ( 15 ), Salmonellosis ( 3 ), Pasteurellosis ( 3 ), Staphylococcus aureus (1, 16) have been reported to worsen the health condition of the affected chicken by Av. Paragallinarum ( 1 , 3 ).

The presence of other bacterial pathogens contributed to the severity and complexity of the disease ( 1 , 12 ). Siddique, SU ( 17 ) in the study of viral and bacterial infection in respiratory distress cases of poultry flocks scattered in some districts of Punjab, Pakistan, which were seropositive and seronegative for mycoplasma gallisepticum using PCR test, reported 5.7% and 3.8% detection rate of Av. Paragallinarum, respectively ( 3 ). In a study conducted in Jimma, Ethiopia the prevalence of 22.4% of IC was reported in backyard chicken of five rural areas by PCR from November 2011 to April 2012 ( 18 ). The intensity of the chicken population and climate of villages seems to have a profound effect on the prevalence of infectious coryza ( 1 , 18 ). IC in neighboring countries of Iran such as Pakistan ( 17 , 19 ), Turkey (20), Iraq ( 21 ), and regional country of India ( 22 , 23 ), and Bangladesh ( 24 ) have also been epidemiologically reported in layer and breeder farms in addition to backyard chicken. Further studies should be conducted to investigate the prevalence of IC among different types of chicken and its possible role in complex respiratory syndrome in Iran.

The variation in antimicrobial sensitivity patterns between isolates from some countries emphasizes the importance of active and continuous monitoring of Av. Paragallinarum isolates ( 25 , 26 ). Limited information exists on isolation and antibiotic susceptibility of Av. Paragallinarum prevalence in Iran, which is limited to the report of Banani, Pourbakhsh ( 5 ) Contrary to his report, the present study alters AST results related to the development of complete antibacterial resistance against oxytetracycline and Lincomycin among Av. Paragallinarum isolates, despite the expected low usage of the antibacterial medications in those chickens.

Extensive use of antibacterial drugs to control secondary bacterial infection following viral diseases particularly immunosuppressive viral infection in commercial flocks imposes great pressure on emerging multi-antimicrobial resistant bacteria including Av. Paragallinarum ( 26 ). In the present study, NAD independent strain of Av. Paragallinarum has not been isolated despite careful examination of culture plates.

Moreover, the antibacterial drug resistance of IC agents may threaten poultry and public health. A more in-depth study is recommended to develop a low-cost autogenous vaccine for backyard poultry to prevent disease and antimicrobial resistance. The present study, to the best of our knowledge, is the first report of IC agent isolation and genetic identification by determining partial hemagglutinin gene sequencing (MN928965-8) from backyard chicken in Iran. Further studies are needed to investigate NAD independent IC strains and unusual growth requirements variants ( 27 ) of Av. Paragallinarum.

Authors' Contribution

Study concept and design: A. N.

Acquisition of data: M. B.

Analysis and interpretation of data: S. Gh. M.

Drafting of the manuscript: A. Sh.

Critical revision of the manuscript for important intellectual content: M. B.

Statistical analysis: A. N.

Administrative, technical, and material support: M. B.

Ethics

Ethical principles of working with laboratory animals in the present study were approved by Education and Research Deputy of the Jihad-Agriculture Ministry, Tehran, Iran under the project number: (NO: 12-18-18-9452-94003).

Conflict of Interest

The authors declare that they have no conflict of interest.

Grant Support

The authors would like to thank the Education and Research Deputy of the Jihad-Agriculture Ministry, Tehran, Iran for financial support of this article (NO: 12-18-18-9452-94003).

Acknowledgement

The authors would like to express their sincere gratitude to Razi Vaccine and Serum Research Institute and the colleagues of the Poultry Diseases Research and Diagnosis Department, especially Ali Chavoshzadeh.

References

1. Blackall PJ, Soriano‐Vargas E. Infectious Coryza and Related Bacterial Infections. In: swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair V, editors. Diseases of Poultry. Wiley-Blackwell: Ames; 2013.
2. Blackall pJ, Soriano‐Vargas E. Infectious Coryza and Related Bacterial Infections. In: David ES, editor. Diseases of Poultry. 2. Wiley-Blackwell: Hoboken, NJ; 2020.
3. Sandoval VE, Terzolo HR, Blackall PJ. Complicated infectious coryza outbreaks in Argentina. Avian Dis. 1994; 38(3):672-8.
4. Bozorgmeri fard MH. Determine the sensitivity of pathogenic bacteria in poultry to different antibiotics. Iran J Vet Med. 1980; 35:101-88.
5. Banani M, Pourbakhsh SA, Khaki P, Goodarzi H, Moazeni-Jula G, Ghodsian N. Isolation, Identification and antibiotic sensitivity of Haemophilus Paragallinarum isolates from commercial layer flocks affected by infectious coryza. [In Persian]. Pajouhesh Sazandegi. 2007; 73:128-35.
6. Blackall, Blackall Pj, Yamamoto r, Infectious Coryza. In: D. E. Swayne JRG, M. W. Jackwood, J. E. Pearson & W. M. Reed. , editor. A Laboratory Manual for the Isolation and Identification of Avian Pathogens 4th ed. Kendal/hunt: American Association of Avian Pathologists; 1998. p. 29-34.
7. Moore D, D. D. Preparation of Genomic DNA from Bacteria. In: Wilson K, editor. In Current Protocols in Molecular Biology: John Wiley & Sons, Inc.; 2002. p. 2.1.-2.1.10.
8. Nouri A, Banani M, Toroqi R. Immunogenicity of Infectious Coryza vaccine against a native isolate of Avibacterium paragallinarum from Iran. Iran Vet J. 2018; 14(4):96-105.
9. Chen X, Miflin JK, Zhang P, Blackall PJ. Development and application of DNA probes and PCR tests for Haemophilus paragallinarum. Avian Dis. 1996; 40(2):398-407.
10. Clothier KA, Stoute S, Torain A, Crossley B. Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites. J Vet Diagn Invest. 2019; 31(5):714-8.
11. Chen X, Song C, Gong Y, Blackall PJ. Further studies on the use of a polymerase chain reaction test for the diagnosis of infectious coryza. Avian Pathol. 1998; 27(6):618-24.
12. Clothier KA, Torain A, Reinl S. Surveillance for Avibacterium paragallinarum in autopsy cases of birds from small chicken flocks using a real-time PCR assay. J Vet Diagn Invest. 2019; 31(3):364-7.
13. Kato K. Infectious coryza of chickens. V. Influence of Mycoplasma gallisepticum infection on chicken infected with Haemophilus gallinarum. Natl I Anim Health Q. 1965; 5(4):183-9.
14. Morales-Erasto V, Falconi-Agapito F, Luna-Galaz GA, Saravia LE, Montalvan-Avalos A, Soriano-Vargas E E, et al. Coinfection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale in Chickens from Peru. Avian Dis. 2016; 60(1):75-8.
15. Paudel S, Hess M, Hess C. Coinfection of Avibacterium paragallinarum and Gallibacterium anatis in Specific-Pathogen-Free Chickens Complicates Clinical Signs of Infectious Coryza, Which Can Be Prevented by Vaccination. Avian Dis. 2017; 61(1):55-63.
16. Kishida N, Sakoda Y, Eto M, Sunaga Y, Kida H. Co-infection of Staphylococcus aureus or Haemophilus paragallinarum exacerbates H9N2 influenza A virus infection in chickens. Arch Virol. 2004; 149(11):2095-104.
17. Siddique A, S U R, I H, G M. Frequency Distribution of Opportunistic Avian Pathogens in Respiratory Distress Cases of Poultry. Pak Vet J. 2012; 32(3):386-9.
18. Dereja I, Hailemichael D. Infectious Coryza in Jimma Backyard Chicken Farms: Clinical and Bacteriological Investigation. J Vet Sci Technol. 2017; 8(1):412.
19. Hasan S, Ahmad K, Fawad N, Siddique B, Rehman H. Current respiratory disease problem and the probes in chicken. Pak Vet J. 2002; 22:17-20.
20. Findik A, Yardimci H. The comparison of agglutination, hemagglutination inhibition and indirect hemagglutination tests in the serological diagnosis of infectious coryza in chickens. In turkish. Ankara Üniv Vet Fak. 2010; 57:69-72.
21. Rashid RA, Pociecha JZ. Epidemiological study of an outbreak of infectious coryza on a poultry farm in Iraq. Avian Dis. 1984; 28(1):235-7.
22. Patil VV, Mishra D, Mane DV. 16S ribosomal RNA sequencing and molecular serotyping of Avibacterium paragallinarum isolated from Indian field conditions. Vet World. 2017; 10(8):1004-7.
23. Patil VV, Mishra D, Mane DV. Virulence pattern of Avibacterium paragallinarum isolates studied from Indian field condition. Int J Livest Res. 2017; 7(2):201-7.
24. Khatun MM, Lijon MB, Islam MA, Sultana N. Detection of antibiotic resistant Avibacterium paragallinarum from broiler chickens in Bangladesh. J Adv Vet Anim Res. 2016; 3(2):173-7.
25. Luna-Galaz GA, Morales-Erasto V, Peñuelas-Rivas CG, Blackall PJ, Soriano-Vargas E. Antimicrobial Sensitivity of Avibacterium paragallinarum Isolates from Four Latin American Countries. Avian Dis. 2016; 60(3):673-6.
26. Nhung NT, Chansiripornchai N, Carrique-Mas JJ. Antimicrobial Resistance in Bacterial Poultry Pathogens: A Review. Front Vet Sci. 2017; 4:126.
27. Blackall PJ, Christensen H, Bisgaard M. Unusual growth variants of Avibacterium paragallinarum. Aust Vet J. 2011; 89(7):273-5.