Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2018 15(3):2583-2590 OPEN ACCESS

First record of avium in Egyptian turkey flocks

Ahmed Erfan1, Jihan Badr2 and Mahmoud Abd-elhalim3

1Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki- Giza, Egypt 2Poultry Diseases Department, Animal Health Research Institute, Dokki- Giza, Egypt 3Brucella department, Animal Health Research Institute, Dokki- Giza, Egypt

*Correspondence: [email protected] Accepted: 04 Aug. 2018 Published online: 30 Sep. 2018 To screen Bordetella avium in Egyptian turkey flocks, tracheal swabs and nasal exudates were gathered from 21 turkey farms out of five Egyptian governorates. Bacteriological examination revealed the isolation of 2 strains. Both isolates displayed different resistance rates regarding the ten applied antibiotics. Both strains agglutinated guinea pig RBCs and adhered to the tracheal rings. Confirmatory recA gene PCR was performed. Moreover, blaTEM, tetA(A), aada1, sul1 and dfrA genes were amplified in both B. avium strains. Partial sequencing of the amplified 740 bp of recA gene revealed 100% maximum identity with German B. avium ATCC 35086 strain and with the American strain 197N.To the best of our knowledge, this is the 1st record of B. avium in Egyptian turkey flocks. Keywords: Bordetella; avium; PCR; sequence; resistance

INTRODUCTION production cycle and the probability of the past Bordetella avium (BA), that cause turkey infection increases while birds get older (Śmiałek bordetellosis, (moreover called turkey coryza) is et al., 2015). The increased incidence of an exceptionally infectious upper respiratory concurrent infections during turkey coryza pathogen that specially infects turkey poults outbreaks contributes to the poor overall leading to high morbidity and minimal mortality performance of B. avium-infected flocks. Antibiotic and is frequently followed by serious secondary treatment of turkey coryza usually fails; likely, infections that lead to great and terrible economic such antibiotics treat secondary infections instead impacts for turkey industry. It hasn’t been of treating B. avium infection. Four toxins are estimated to be transmitted vertically. But, the typically produced by B. avium: endotoxin, survival of BA for at least 6 months in intact damp tracheal cytotoxin, heat-labile dermonecrotic toxin litter has been reported (Jackwood and Saif, and osteotoxin (Rimler, 1985). Deformation of 2008), which contributes to epidemiological tracheal rings and damage to articular cartilages situation of Bordetella avium infection in turkeys. that are usually observed in field cases of B. A review in 2009 ranked B. avium as the third avium infections may be related to osteotoxin infectious pathogen in a list of the most activity (Yersin et al., 1991). B. avium adheres to threatening health issues for the American turkey tracheal epithelium to colonize the turkey’s industry as reported by poultry stakeholders breathing tract (Arp et al.,1988; Temple et al., (Clark et al., 2011). B. avium infection was 2010). Hemagglutinin produced by B. avium in described as a common infection among turkey addition to autotransporter proteins and fimbriae flocks that is frequently reported during the whole play the main role in this process (Moore et al., Erfan et al., Bordetella avium in Egyptian turkeys

1994; Loker et al., 2011; Stockwell et al., 2011). Isolation of B. avium. Interestingly, the fimbrial locus of B. avium is Isolation of the microorganism from samples regulated in response to temperature (37oC), and was performed following previously validated the bacterium is able to adhere to the host’s protocol (Dufour-zavala et al., 2008). The pooled respiratory epithelium only under such conditions, samples were cultured in blood agar base with 5% this was proved by testing the expression of fimA- of defibrinated sheep blood and in MacConkey D by real time PCR, when FimA gene was only agar at 37°C under aerobic conditions for 24-48 h. expressed at 37oC, however, expression failed at After the incubation duration, colonies displaying temperatures below 25oC (Loker et al.,2011). suggestive morphology (pinpoint, convex and Even though B. avium is thought to be strictly an glistening with a pearl to gray color) were picked animal pathogen that causes tracheobronchitis in up and subjected to phenotypic characterization. birds, human cases of respiratory disorders related to B. avium have been recorded lastly in Phenotypic characterization. patients with (Spilker et al., 2008), The suspected colonies were identified thereby demonstrating that B. avium and B. microscopically and biochemically as formerly avium-like organisms are opportunistic human described (Dufour-zavala et al., 2008; Quinn et pathogens. al., 1994). Employing 16S rRNA gene sequencing, two isolates had been identified from human Antimicrobial susceptibility. that showed 98% and100% nucleotide Antibiogram check was performed following similarity to the B. avium ATCC 35086 strain the Kirby Bauer approach, compliant with the (Harrington et al., 2009). standardized protocol established by the A primary barrier to the improvement of documents VET01-A4 and VET01-S2 (CLSI, advanced therapy against B. avium infections is 2013) using 0.5 McFarland standard adjusted that very little is known about this bacterium or colony at log phase of growth (suspended in about the way by which it infects birds to cause Mueller-Hinton broth), zones of inhibition were disease. However, a major breakthrough in measured around the antimicrobial disks and then understanding the biology of B. avium is rising, interpreted (CLSI, 2013).The used antimicrobials because the genome sequence of a were ampicillin 10 mcg (AMP), cefotaxime 30 mg representative strain of B. avium was successfully (CTX), norfloxacin 10 mg (NOR), ciprofloxacin 10 generated and analyzed. This has discovered the mg (CIP), erythromycin (E), florfenicol 30 mg complete genetic code of the bacterium (Sebaihia (FFC), gentamycin 10 mg, lincomycine (L), et al., 2006). sulphamethoxazole/trimethoprime (SXT) and The target of the present work was the oxytetracycline 30 mg (OT). isolation, identification, molecular characterization and pathogenicity testing of B. avium from turkey assay of B. avium. field cases suffering from respiratory troubles. Hemagglutination assay.

A hemagglutination assay using guinea pig MATERIALS AND METHODS erythrocytes was conducted as previously Collection of samples. described (Dufour-zavala et al., 2008). Strains Tracheal swabs and nasal exudates have were grown on brain heart infusion agar at 37°C been collected aseptically from 10 poults in each for 36-48 h. Bacterial growths were washed from the plates with phosphate buffer saline (PBS) and farm out of 21 turkey farms. Sampling was 9 focused on birds showing respiratory diluted to a concentration of approximately 5 x 10 manifestations. Sampled farms represented 5 cell/ml (0.5 optical density at 600 nm). Equal Egyptian governorates that harbor substantial volumes of bacterial suspension and erythrocytes turkey breeding (Giza, Gharbeya, Menofeya, suspension (2% packed-cell volume in PBS) were Sharkeya and Faium). Samples had been mixed on a glass slide and observed for transported in a cooler to the laboratory within 1-3 agglutination after gentle rocking. h. After pooling of the samples (samples from Tracheal attachment assay. each farm were pooled as one sample), they have A tracheal attachment assay was applied as been subjected to bacteriological and molecular formerly described (Temple et al., 1998). identification. Transverse 2 mm length tracheal rings were

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Erfan et al., Bordetella avium in Egyptian turkeys aseptically obtained from 26 days-old turkey purified by Centrisep spin column. DNA embryos after decapitation. Three tracheal rings sequences were performed by using 3130 genetic were incubated in separate wells containing EBSS analyzer (Applied Bio systems, HITACHI, Japan). culture medium (Sigma Chemical Co., St. Louis, Sequence homology to GenBank accessions was Mo.) and a 0.5 ml of B. avium broth culture (of established through BLAST ®analysis (Altschul et about 2 x 107 /ml) ,it was then incubated al., 1990). The sequence identities were at 42oC for 3 h. on a shaker incubator until determined by MegAlign module of Lasergene examined by inverted microscope. DNAStar (Thompson et al., 1994) and phylogenetic analyses was performed using Polymerase chain reaction. maximum likelihood, neighbor joining and DNA was extracted from culture samples maximum parsimony in MEGA6 (Tamura et al., using QIAamp DNA Mini kit (Qiagen, Germany, 2013). GmbH). Unique PCR primers have been supplied from Biobasic (Canada) as shown in Table (1). RESULTS AND DISCUSSION PCR reactions were carried out in a 25-μl master mix reactions containing 12.5 μl of Emerald Amp Clinical signs. Max PCR master Mix(Takara, Japan), 1 μl of each B. avium survives for many months on litter and in primer (20 pmol conc.), 7.5 μl of PCR grade water drinking water systems, it’s so difficult to eliminate and 3 μl of template DNA. PCR was performed in the microorganism from the turkey’s environment Applied biosystem 2720 thermal cyclers. PCR (Jackwood and Saif, 2008) and it’s very easy to products were inoculated in agarose gel (Lonza), spread from an infected flock to a healthy one. and Generuler 100 bp ladder (Fermentas, Infected turkeys suffer from mouth respiration, Thermo) was used to check fragments sizes. The oculonasal discharge, submaxillary oedema and gel images had been captured through a gel collapse of the trachea. The same clinical documentation system (Alpha Innotech, Biometra) symptoms had been described by previous and the images were analyzed through the usage studies (Jackwood et al., 1995; Stenzel et al., of laptop software (Automatic Image Capture, 2017). Protein Simple, USA). All of the 21 sampled flocks showed moderate Partial sequencing of recA gene for 2 strains morbidity with variable clinical signs as sinusitis, was done using specific primers. PCR products clear nasal discharge, foamy-watery eyes, had been purified by QIAquick PCR Product coughing, mouth breathing, dyspnea and tracheal extraction kit (Qiagen, Valencia). Sequence was rales; some flocks showed marked altered performed by Bigdye Terminator V3.1 cycle vocalization. sequencing kit (Perkin-Elmer), and then it became

Table 1: Oligonucleotide primers used in the study

Target gene Oligonucleotide sequence Annealing Amplified Reference (5´- 3´) temperature (˚C) fragment (bp) recA CCGCTTATGCATGACCGTTT 60 740 Stenzel et al., 2017 TTCAGGGTTGCCGAACATCA blaTEM ATCAGCAATAAACCAGC 54 516 Colom et al., 2003 CCCCGAAGAACGTTTTC Aada1 TATCAGAGGTAGTTGGCGTCAT 54 484 Randall et al. 2004 GTTCCATAGCGTTAAGGTTTCATT TetA(A) GGTTCACTCGAACGACGTCA 50 576 CTGTCCGACAAGTTGCATGA Sul1 CGGCGTGGGCTACCTGAACG 60 433 Ibekwe et al., 2011 GCCGATCGCGTGAAGTTCCG dfrA TGGTAGCTATATCGAAGAATGGAGT 60 425 Grape et al., 2007 TATGTTAGAGGCGAAGTCTTGGGTA

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Figure 2. Phylogenetic tree for recA partial sequences that was generated using maximum likelihood, neighbor joining and maximum parsimony in MEGA6. Phylogenetic tree showed clear clustering of the 2 isolated Egyptian strains with the 2 B. avium strains uploaded from gene bank.

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Figure 3. Sequence distance created by the MegAlign module of Lasergene DNAStar. Sequence identities of 100% homology were recorded between two Egyptian B. avium sequenced strains and 2 B. avium strains uploaded from gene bank public data.

as Proteus spp., Pseudomonas spp. and E. coli Bacteriological identification. which may have overgrown the BA colonies and Bacteriological examination of samples made it difficult to isolate the microorganism in resulted in the isolation of two BA strains (4.76%) pure form. Moreover, as the disorder is highly out of the 21 tested samples. Colonies were contagious and infection disperse readily through motile gram negative, non-fermentative, strictly flocks, the damage brought to the airway of aerobic coccobacilli (pinpoint, convex and turkeys by means of B. avium increases the glistening with a pearl to gray color). Both strains tendency of those birds to different respiratory showed positive reactions for oxidase, infections. Also, the random use of antibiotics and citrate; while, they exhibited negative results might have decreased the isolation rate. for and nitrate reduction. On triple sugar Both isolates showed an antibiotic resistance iron agar (TSI), no acid production in the butt or profile that was so close to that obtained by the slant was determined after overnight former studies (Mortensen et al., 1989) when the incubation, however, H2S production was antimicrobial susceptibility of B. avium and B. observed. bronchiseptica isolates were reported to be Antibiogram of the isolates towards ten resistant to tetracycline, lincomycin and different antibiotics showed that norfloxacin, sulfadimethoxine. However, it has been formerly ciprofloxacin, cefotaxime, florfenicol and to a reported that tetracycline resistance has been lesser extent gentamicin (for only one strain) associated with a limited number of B. avium exposed the highest effectiveness towards both strains (Cutter and Luginbuhl, 1991). On the Bordetella isolates, respectively. While, ampicillin, other hand, other authors (Beach et al., 2012) erythromycin, oxytetracycline, tested 17 B. avium strains collected over thirty sulphamethxazole/trimethoprime and lincomycin years and stated that all tested isolates were exhibited the highest resistance rates. sensitive to gentamicin, cefoperazone, cefepime, Although, this becomes the 1st record of B. ceftazidime, piperacillin, and amikacin. avium isolation in Egyptian turkey flocks, the low In another study (Malik et al., 2005), four B. isolation prevalence can be related to the avium strains were isolated in Minnesota in 1998– existence of different contaminant microorganisms 1999 (among others) and were sensitive to both

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Erfan et al., Bordetella avium in Egyptian turkeys ampicillin and tetracycline. While, they were dermonecrotic toxin (DNT) and tracheal cytotoxin resistant to erythromycin. On the other hand, via many Bordetella species and proved that they among 50 B. avium strains, the range of were concerned in the damage of tracheal tissue resistance against cephalosporin, penicillin, and concluded that the capability of a strain to erythromycin, and enrofloxacin were >50%, but cause an in vivo illness is correlated with its <25% for all other examined antimicrobials capability to adhere to the ciliated tracheal cells in (Nhung et al., 2017). The turkey isolates showed vitro. wide spectrum of antimicrobial resistance. This is most probably related to the uncontrolled Polymerase chain reaction. application of antimicrobials in reared flocks. So, As shown in Figure 1, Bordetella avium recA increasing awareness about the use of gene specific PCR products had been detected in antimicrobials for treatment of poultry pathogens two farms (The 2 cases were confirmed by will decrease treatment failure, and reduce related microbiological isolation). On the other hand, 516 financial losses. bp, 576 bp, 484 bp, 433 bp and 425 bp for blaTEM, tetA(A), aada1, sul1 and dfrA genes, Virulence assays of B. avium. respectively were amplified in both B. avium Both harvested strains agglutinated guinea detected strains. The positive results encountered pig RBCs; as B. avium Hemagglutinin is a for the antibiotic resistance genes additionally crucial virulence element (Moore et al., 1994), the agreed with the revealed profile by disk diffusion encountered findings proved the great virulence of method. the 2 isolated strains. Through a related study (Rumińska and Koncicki, 1999), it was suggested DNA sequence. that unique strains of BA are recognized to DNA sequencing for 740 bp of recA gene from produce toxins as endotoxin, tracheal cytotoxin, 2 strains have been generated, then submitted heat-stable dermonecrotic toxin and osteotoxin as (GenBank accession numbers MG821490 and well as hemagglutinin that are concerned in MG821491). As shown in figures 2 and 3, partial pathogenesis of the disease. Also, the findings sequencing of the amplified 740 bp of recA gene agreed with the preceding study that stated that B. revealed a hundred % maximum identity (among avium agglutinates guinea pig erythrocytes via an each others) and with ATCC 35086 strain unknown mechanism and loss of (Genbank accession AY124330.1) (Gentry-Weeks hemagglutination capability results in attenuation et al., 1988), that had been harvested from upper (Temple et al., 2010). respiratory tract of diseased turkey in Germany. Regarding the tracheal attachment assay, The isolated strains showed additionally one tracheal rings had marked damage, with the hundred % maximum identity to the American disruption of the mucosal architecture. strain 197N (Genbank accession AM167904.1) Microscopic observation revealed cilia damage at that was harvested from a diseased turkey in a some areas 4 h. post culturing. commercial flock in the early 1980s in Ohio In this study, we utilized embryonic tracheal (Sebaihia et al., 2006). rings (rather than rings from live poults), as Maximum identity to other Bordetella strains embryonic rings were easily obtained aseptically (other than from B. avium) ranged from 87.3% (B. and show uniform, reproducible results. Moreover, pertussis) to 89.7% (B. hinzii). providing an organ that is matching to the in vivo colonization site makes the in vitro and in vivo CONCLUSION results more closely comparable. The obtained The 1st record of B. avium in Egyptian turkeys results for the tracheal attachment assay became encountered that represent a significant confirmed that the two isolated strains were highly trouble to the turkey industry, inflicting poor flock virulent. performance and increasing the incidence of other The revealed results in this work are in great infections. Hence, extensive clean up measures concordance with a preceding study (Gentry- are required to remove the bacterium from Weeks et al., 1988) where B. avium was contaminated premises, along with complete examined for virulence factors together with the elimination of all litter, and disinfection of all filamentous hemagglutinin, and tracheal cytotoxin, surfaces, feeders and drinking water systems. and concluded that a dermonecrotic toxin and Prevention of infection calls for strict tracheal cytotoxin are the main putative virulence biosecurity measures. Antibiotic treatment elements of B. avium. Morever, they detected of turkey coryza has variable success; it is likely

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Erfan et al., Bordetella avium in Egyptian turkeys that antibiotics in fact deal with secondary CLSI, Wayne, PA, USA. infections instead of B. avium infection. Colom K, Pèrez J, Alonso R, Fernández-Aranguiz A, Lariňo E, Cisterna R., 2003. Simple and CONFLICT OF INTEREST reliable multiplex PCR assay for detection of The authors declared that present study was blaTEM, blaSHV and blaOXA-1 genes in performed in absence of any conflict of interest. . FEMS Microbiol Lett 223(2):147-151. ACKNOWLEGEMENT Cutter DL, LuginbuhI GH., 1991. Characterization The authors wish to thank Animal health research of Sulfonamide Resistance Determinants and institute high management for funding this Relatedness of Bordetella avium R . research and their support regarding reagents and 26(2):136–140. equipment supply during all the study steps. Dufour-zavala L., Swayen DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW and AUTHOR CONTRIBUTIONS Woolcock PR., 2008. Bordetellosis. IN: A All authors contributed equally in all parts of laboratory manual for the isolation, this study. identification and characterization of avian Copyrights: © 2017 @ author (s). pathogens, 5th ed., Athens: American This is an open access article distributed under the Association of Avian Pathologists 1-249. terms of the Creative Commons Attribution License Gentry-Weeks CR, Cookson BT, Goldman WE, (CC BY 4.0), which permits unrestricted use, Rimler RB, Porter SB, Curtiss R 3rd., 1988. distribution, and reproduction in any medium, Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella provided the original author(s) and source are avium. Infect Immun 56(7):1698–1707. credited and that the original publication in this Grape M, Motakefi A, Pavuluri S, Kahlmeter G., journal is cited, in accordance with accepted 2007. Standard and real-time Multiplex PCR academic practice. No use, distribution or methods for detection of trimethoprim reproduction is permitted which does not comply resistance dfr genes in large collections of with these terms. bacteria. ClinMicrobiol Infect 13(11):1112– REFERENCES 1118. Altschul SF, Gish W, Miller W, Myers EW, Lipmanl Harrington AT, Castellanos JA, Ziedalski TM, DJ., 1990. Basic Local Alignment Search Clarridge JE 3rd, Cookson BT., 2009. Tool. J Mol Biol 215(3):403-410. Isolation of Bordetella avium and novel Arp LH, Leyh RD, Griffith RW., 1988. Adherence Bordetella strain from patients with of Bordetella avium to tracheal mucosa of respiratory disease. Emerg Infect Dis turkeys: correlation with hemagglutination. 15(1):72–74. Am J Vet Res 49(5):693-696. Ibekwe AM, Murinda SE, Graves AK., 2011. Beach NM, Thompson S, Mutnick R, Brown L, Genetic Diversity and Antimicrobial Kettig G, Puffenbarger R, Stockwell SB, Resistance of from Human Miyamoto D, Temple L., 2012. Bordetella and Animal Sources Uncovers Multiple avium Antibiotic Resistance, Novel Resistances from Human Sources. PLoS Enrichment Culture, and Antigenic ONE 6(6):e20819. Characterization. Vet Microbiol 160(1- Jackwood MW, McCarter SM, Brown TP., 1995. 2):189–196. Bordetella avium: an opportunistic pathogen Clark SR, Corsiglia C, Bailey A., 2011. Current in Leghorn chickens. Avian Dis 39(2):360- Health and Industry Issues Facing the 367. Turkey Industry. Published in the 2011 Jackwood MW, Saif YM., 2008 Bordetellosis Annual Proceedings of the USAHA meeting; (turkey coryza). In: Saif YM (ed) Diseases of Transmissible Diseases of Poultry and Other Poultry 12th edition. Blackwell Publishing, Avian Species Committee. Ames;774-788. Clinical and Laboratory Standards Institute, Kersters K, Hinz KH, Hertle A, Segers P, Lievens Performance standards for antimicrobial disk A, Siegmann O, De Ley J., 1984. Bordetella and dilution susceptibility tests for bacteria avium sp. nov., isolated from the respiratory isolated from animals; (VET01-A4: Approved tract of turkeys and other birds. Int J Syst standard—fourth edition) and (VET01-S2: Bacteriol 34(1):56-70. Second informational supplement). 2013. Loker SB, Temple LM, Preston A., 2011. The

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