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Detection and Molecular Characterization of Avibacterium paragallinarum from Poultry
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Methods: A total of 128 samples (viz., heart blood swabs, tracheal, eye and infra orbital swabs) suspected for infectious coryza were screened for the presence of Av. paragallinarum by isolation and molecular characterization. Information regarding farm/flock strength, age of birds, method of rearing, production performance, clinical symptoms manifested and mortality patterns from various organized poultry farms were recorded. A total of five isolates of Av. paragallinarum have been recovered from suspected materials processed for isolation. Two isolates were sequenced and analysed.
Result: All the isolates showed same biochemical characters but exhibited variability in the fermentation of mannose, mannitol and sorbitol. The nucleotide variation of Av. paragallinarum present in Indian subcontinent differs in their virulence and pathogenicity. The study was also focused for the molecular characterisation of Avibacterium species from different isolates. Though the clinical signs and gross pathological signs were similar, the nucleotide variation between isolates was observed. Sequencing of ‘hagA’ gene showed that the two isolates were closely related to the strains of China, Australia, Mexico and India which shared 94-100% homology.
Infectious coryza is one of the major problems affecting commercial poultry industry, in India. In India, the first incidence of Infectious Coryza in India was reported in 1958 (Adalkha, 1967). The laboratory diagnosis of IC caused by Av. paragallinarum infection is based mainly on demonstration and confirmation by conventional methods such as isolation and identification of the organisms.
Characterization of Indian isolates of Av. paragallinarum by Page serotyping Scheme has shown that, serovar A and serovar C were prevalent in India but no isolate of serovar B was identified (Tongaonkar et al., 2002). Similarly, Patil et al., (2016) found serovar B for the first time in India while serovar C was more prevalent followed by A than B. Later, Patil et al., (2017) also found serovar A, B and C by molecular typing. Contrarily, Anne et al., (2022) reported higher prevalence of serovar B than A while there was no serovar C in their study. Since infectious coryza is one of the major problems detected in different parts of India affecting commercial poultry industry causing significant economic loss that emphasizes the need for molecular diagnosis and pathological studies of Av. paragallinarum serovars circulating in India.
Though few studies on identification of Av. paragallinarum is available from India, very scanty literature is available from the state of Tamil Nadu. To the best of our knowledge, little is known about the molecular diagnosis of complicated cases of infectious coryza in the state of Tamil Nadu, India and very scarce systemic pathogenicity studies have been conducted on Av. paragallinarum. Keeping in view of the above facts, the present study was conducted for isolation, identification and biochemical characterization of Av. paragallinarum from suspected clinical and post-mortem cases of Infectious Coryza. Diagnosis of Infectious Coryza by using gene specific Polymerase chain reaction (PCR) and Pathogenicity studies in chicken were also carried out.
MATERIALS AND METHODS
A total of 128 samples were collected from infectious coryza suspected 73 farms of 85 poultry flocks as well as samples available with Poultry Disease Diagnosis and Surveillance Laboratory, Namakkal, Tamil Nadu. Information regarding farm/flock strength, age, method of rearing, production performance, clinical symptoms manifested and mortality patterns from various organized poultry farms were recorded. The collected samples comprising of heart blood swabs, tracheal, eye and infra orbital swabs. The collected specimens were transported in Ames transport medium and stored in refrigerator till further processing.
Sampling from live birds
Ailing birds with history of respiratory disease and showing signs of infectious coryza viz., swollen infra-orbital sinuses, serous to mucoid oculo-nasal discharge, eye and facial swelling, were selected and gentle pressure was exerted on each infraorbital sinus to flush out mucus through nostrils and sampled using sterile disposable cotton swabs.
Sampling from autopsied birds
Samples were taken from birds either already dead or after scarifying by decapitating the birds showing clinical signs of coryza. The head was swabbed with 70% alcohol and allowed to dry. The mucoid fluid from the infra orbital sinus and nasal or conchal cavities was collected with a nichrome loop.
Primary isolation of NAD-dependent Av. paragallinarum
Blood agar (5-7% v/v sheep blood) with haemolytic Staphylococcus sp.as feeder culture was used as culture media for preliminary isolation of Av. paragallinarum from suspected samples. Chocolate agar with 0.01% (w/v) NADH and Brain Heart Infusion broth were used for further sub culturing and studying growth characteristics as well as purification of genomic DNA. Collected samples were streaked directly onto the chocolate and blood agar plates. The plates were incubated at 37°C for 48 hrs in a traditional candle jar supplying with 5-10% CO2.The colonies grown on Blood agar and Chocolate agar at 37°C after 14 hrs of incubation in 5% CO2 were examined by Gram’s staining. The organism was identified as per Cowan and Steel’s Manual for Identification of Medical Bacteria (Cowan, 1974). All the isolates were subjected to catalase, oxidase, nitrate reduction, indole production and urea hydrolysis tests.
All the isolates, were inoculated into 5-7 days old embryonated chicken eggs via yolk sac route and inoculated eggs were kept at 37°C for 48 hrs. Embryos dying after 24 hrs of inoculation were considered as infected. After 48 hrs the yolk fluid was harvested, dispensed in screw capped 10 ml glass bottles and stored at -20°C for further use.
Genomic DNA was isolated from the overnight grown bacterial culture by using Genomic DNA purification Kit (Promega, USA) and stored at -20°C until use.
Polymerase chain reaction
Initial PCR screening was carried out by using the primer pair targeting the hpg-2 gene of Av. paragallinarum N1 (5'-TGAGGGTAGTCTTGCACGCGAAT-3') and R1 (5'-CAAGGTATCGATCGTCTCTCTACT-3') to amplify specific 500bp fragment (Chen et al., 1998). The amplification was carried out using PCR master mix (2X) (Thermo scientific, USA), with an initial denaturation at 94°C for 4 min, followed by 35 cycles at 94°C for 1 min, 63°C for 1 min, 72°C for 1 min and a final extension at 72°C for 10 min. For sequencing further, the hagA gene was amplified using primer pairs HA1 (TGT AGC TCAAGC AGC TCC ACA AG) and HA2 (TCA AGC GAT AAG TGC TTT ACG ACC) to amplify 900-bp fragment (Rhonda et al., 2002). The amplification was carried out using PCR Master Mix (2X) (Thermo scientific, USA), with an initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 46°C for 1 min, 72°C for 1.5 min and a final extension at 72°C for 10 min. PCR products were purified employing ExoSAP-IT ® (Affymetrix, USA) and sequenced commercially.
The nucleotide sequence data generated were edited and aligned using Sequencing Analyses Software v5.3 (Applied Biosystems, USA) and MEGA 6 software. Reference sequences were downloaded from GenBank database. Phylogenetic analyses and evolutionary associations were inferred in MEGA 6.0 using the Maximum Likelihood algorithm with Kimura-2P correction and 1000 bootstrap replications.
Experimental pathological studies of Av. paragallinarum
Pathogenicity of field isolates of Av. paragallinarum was studied in experimentally infected 7 weeks old chicken. The birds were examined daily for 7 days for clinical symptoms of Infectious coryza, mortality and progression of diseases. The experimentation was performed after obtaining IAEC and IBSC permission (IAEC approval no. 4/2015, dt. 07.07.2015, VCRI, Namakkal and Lr. No. 1675/DFBS/IBSC/IBSC/2015, dt. 04.09.2015, the Dean, faculty of Basic Sciences, MVC, Chennai).
Preparation of inoculum
The inoculum for the experimental infection of birds was prepared according to Reid and Blackall (1984) and Sobti et al., (2001) with slight modifications. Pure bacterial colonies grown on chocolate agar plates were picked and inoculated into Brain Heart infusion broth in test tubes. The test tubes were then incubated for 48 hr under reduced O2 tension. Cultural suspension of 1011 CFU of each isolate was used as the inoculum for pathogenicity tests.
Infection of birds
A total 90 birds were randomly divided into 6 groups (15 birds each group). Group1-5 was infected with one isolate each (Isolates 1 to 5) of Av. paragallinarum. Group-6 were kept as unchallenged control.
Method of inoculation
An inoculum of 0.2 ml of each isolate with viable organisms was inoculated to susceptible seven-week-old chicks through intranasal and infraorbital sinus route for three days.
Post infection observation and Re-isolation of Av. paragallinarum
All the experimentally infected birds were daily observed for clinical symptoms and progression of diseases for 7 days. After the development of clinical signs of infectious coryza, the birds were scarified and decapitated for observation of post-mortem lesions. Tissue samples were collected and the organisms were re-isolated.
RESULTS AND DISCUSSION
All the isolates produced similar reactions in biochemical tests as presented in the Table 1 and Fig 4, 5, 6.
Carbohydrate fermentation tests
All the Av. paragallinarum isolates tentatively identified by biochemical tests were further characterized by various sugar fermentation tests. All the five field isolates failed to ferment Arabinose, Cellobiose, Dulicitol, Galactose, Raffinose and Trehalose, while these isolates showed acid production with Glucose, Sucrose and Fructose without formation of gas. There was variation in acid production from Mannitol, Mannose and Sorbitol. The results of carbohydrate fermentation tests are presented in Table 1.
The overall isolation of Av. paragallinarum in this study was 3.9 per cent (5/128). Infra orbital samples showed highest isolation of 9.1 per cent (3/33) followed by tracheal swabs 4.1 per cent (1/24) and eye swab 1.5 per cent (1/67). Among samples collected from affected birds, the isolation was high in birds below 20 weeks of age (5%) followed by layer chicken 20-40 weeks (2.1%).
The results of the Av. paragallinarum isolates obtained from various samples are furnished in Table 2 and age wise incidence is furnished in Table 3. Similar findings were reported by Khatun et al., (2016), who isolated and characterized Av. paragallinarum from commercial broiler chickens in Bangladesh, during field outbreaks using the bacteriological culture of nasal and ocular discharges, tracheal swab, tracheal wash and infraorbital sinus exudates.
Pathology of spontaneous cases of Infectious coryza
Chickens affected with infectious coryza in most of the natural outbreaks showed serous to mucopurulent oculo-nasal discharges with respiratory rales, conjunctivitis, facial oedema, unilaterally or bilaterally swollen infraorbital and nasal sinuses including eyelids (Fig 7). Foamy lacrimation and purulent exudates sticked to eyes due to fibrino-purulent conjunctivitis. There was drop in egg production and mortality patterns varied with secondary pathogens in environment.
In the early stages of disease, unilateral or bilateral swelling of periorbital region and conjunctivitis and in advanced cases there were closure of eyes, bilateral infraorbital swelling with mucopurulent oculo-nasal discharge, oedema of wattles and intermandibular region. Catarrhal exudates with necrotic debris was found in upper larynx and nasal cavities (Fig 8). Abdominal air sacs were thickened with yellowish cheesy exudates. Ovaries were congested, softened and ruptured yolk leading to egg yolk peritonitis.
Experimental pathological studies
The five group of experimentally infected birds were examined in detail at regular intervals of time for clinical signs and pathological changes and the results were compared with those obtained from spontaneously infected cases. All the infected birds exhibited oedematous swelling of left side of infraorbital sinus with secretion of watery nasal discharge after 24 hrs of infection (Fig 9) and swelling of face and infraorbital sinuses of other side after 48 hrs of inoculation (Fig 10). Some birds showed bilateral swelling of face and infraorbital sinuses, conjunctivitis, serous to mucoid nasal discharge with foul smelling, foamy lacrimation and induration of face after 3 days of infection. Respiratory rales, clear watery diarrhoea and swelling of comb and wattles were noticed after 4 days of infection. After 5 days of infection disappearance of nasal discharge and lacrimation and cessation of diarrhoea were observed but the birds still suffered from swelling of face and wattle after 7 days. Two isolates (PDDSL-1 and 3) also caused mortality (Fig 11) with clinical symptoms. The birds were sacrificed after 3rd day of inoculation for observation of post-mortem lesions and collection of nasal and sinus exudates for re-isolation of organisms. Other birds were kept for observation up to 10 days of post infection. Unchallenged group of birds did not reveal any clinical signs of infectious coryza and also did not show any gross and histopathological lesions of infectious coryza. Anjaneya et al., (2013) and Xu et al., (2019) observed less severe ocular discharges and mild swelling of infraorbital sinus in chicken after experimental infection when compared to other reports. Further they also reported that this could be due to the bacterial strain, pathogenicity and host susceptibility. Anjaneya et al., (2013) demonstrated the infection of Av. paragallinarum in chickens after multiple infusion through direct intra-sinus inoculation of birds over a period of three days and development of clinical signs.
The nasal cavities were filled with greyish white thick exudates and mucus membrane of nasal cavities showed congestion and oedematous swelling after 2 days of infection. Infraorbital sinuses cavities were filled with greyish white watery exudate and oedema of subcutaneous tissue of periorbital region, mucus membrane showed hydropic swelling after 3 days of infection. Larger amount of yellowish cheesy exudates was noticed after 5 days of infection. Trachea revealed mild congestion of mucus membrane.
Molecular detection of Avibacterium paragallinarum
Isolation and identification of Av. paragallinarum is difficult and the organism is so fragile the rate of isolation will always be lesser hence, several incidences molecular diagnostic assay are performed (Muhammad and Sreedevi, 2015). Similar to this study Anne et al., (2022) reported isolation of 5 Av. paragallinarum and also reported the usefulness of PCR over isolation. PCR performed on all the five isolates of Av. paragallinarum revealed an amplified product of approximately 500 bp amplicon with hpg-2 PCR and 900 bp amplification with hagA PCR (Fig 12). Similar findings were reported by Muhammad and Sreedevi (2015), using the PCR technique, detected Av. paragallinarum from outbreaks of infectious coryza in Andhra Pradesh of India.
Sequencing and sequence analysis of ‘hagA’ gene
Sequencing and BLAST analysis of ‘hagA’ of two isolates (PDDSL 1 and 2) of Av. paragallinarum indicated that the isolates were closest relationship with Av. paragallinarum strains of China, Australia, Mexico and India which shared 94-100% homology (Fig 13).
Zhang et al., (2016) showed that the strains circulating in China before 2014 and after 2015 had different ancestors and that new strains found in China were derived from earlier Indian strains, with some genetic differences.
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