PREVALENCE, PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF GALLIBACTERIUM ANATIS ISOLATED FROM LAYER.

Ashraf A. Abd El Tawab 1 , Waffaa M. Hassan 2 and Mariam M. El Shemy 3 . 1. Bacteriology, Mycology and Immunology Department Faculty of Veterinary Medicine Benha University. 2. Reference laboratory for quality control on poultry production. 3. Animal Health Research Institute ,Tanta branch. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 18 October 2018 Final Accepted: 20 November 2018 Published: December 2018

Gallibacterium species are Gram-negative rod-shaped or pleomorphic disterputed singly or in pairs and non-motile. Colonies are grayish and non-transparent with strong hemolytic zone on Sheep blood agar, but eventually translucent at the periphery with a butyrous consistency, circular, raised with an entire margin smooth, shiny, and 1-2 mm in diameter at 37 o C for 24-48 hrs. nonspore forming. Grow under microaerophilic condition. positive for Catalase, oxidase, and phosphatase but negative for indole and urea (Christensen et al.,2003a). Prevalence proportions of haemolytic G. anatis was highly influenced by the production system and the biosecurity levels .
G. anatis F149T has ability to express fimbriae which responsible for mucosal attachment and colonization to oropharyngeal epithelial cells (Salgado-Lucio et al.,2012).
G .anatis is able to produce a cytolytic RTX-toxin as a virulence factor which responsible for hemolytic phenotype and leukocytic activity. (Kristensen et al.,2010).
Identification of Pasteurellaceae members by traditional methods are difficult in culturing, isolation and weak reactions toward some of the phenotypic tests used for identification (Christensen et al.,2003) Consequently, the aim of the present study was to apply a PCR specific for G.anatis to allow accurate and unambiguous identification, where G.anatis had a relatively short internal transcribed 16S to 23S rRNA gene sequence compared to other members of Pasteurellaceae and to focus on its virulence and resistance genes.
Materials And Methods:collection of samples:-50 egg laying chickens (recently dead and diseased) suffering from peritonitis, salpingitis and decreased egg production were collected from Al-Gharbia Governorate in the period of (2016-2017) samples from (trechea, ovary and oviduct swabs) were aseptically collected and transferred immediately in ice box to the laboratory.

Bacteriological examination of the samples:-
The samples were identified on the basis of Gram staining after streaking into media as: Columbia blood agar, Sheep blood agar, MacConky's agar and Dextrose starch agar media (oxoid).
After cultured on media incubated at 37 o C for 24 hours with 7.5 %CO2.
Each colony has shown a typical colonial appearance was subjected to staining by Gram's and giemsa stain. Biochemical identification was examined for catalase, oxidase, indole and urea tests. according to Christensen et al.,2003a.

Congo red binding test:-
Congo red binding test was conducted for detection of virulence G. anatis according to Berkhoff and Vinal,1986.

Extraction of DNA and PCR Conditions:-
The isolates were cultivated on 5% citrated bovine blood agar overnight. A single colony was incubated at 37 • C on brain heart infusion broth. Both DNA extraction and Emer-ald Amp GT PCR master mix preparations were conducted using commercial kits (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. (Sambrook et al., 1989).
Antimicrobial suscessptabilty test for Gallibacterium anatis:-G. anatis isolates were cultured on blood agar plate containing 10 % sheep blood. The colonies were scrapped out and re-suspended in Brain heart infusion broth to a turbidity of 0.5 McFarland scales. One hundred microlitres of the culture was swabbed onto the brain heart infusion agar according to Bauer et al., 1966 then the following antibiotic discs erythromycin(15µg), gentamicin (10µg), doxycycline (30µg), oxytetracyclin (15µg), ciprofloxacin (5µg), cefotaxime (30µg), sulfadimethoxine trimethoprim (25µg) and amoxicillin|clavulinic acid (30 µg) were dispensed on media and incubated over night at 37° C under microaerophilic condition. inhibition zones were interpreted according to the criteria recommended by the Clinical and Llaboratory Standerds Iinstute (CLSI,2011).

Detection of resistance and virulence genes:-
The different primers used in this study are described in Table (1).

Incidence of G.anaitis in layer chickens:-
In the present study out of 50 egg laying chickens suffering from peritonitis, salpingitis and decreased egg production 8/50(16%) were positive for G.anaitis isolation. Clinically examined birds showed signs of restlessness, ruffled feathers and drop in egg production. P.M lesions were, septicemia, peritonitis, salpingitis and degeneration in egg follicle .The recovered isolates were iridescent on Dextrose Starch Agar medium and show concentric rings. Sub-culturing on MacConkey's agar showed no growth. Colonies of G.anatis on Blood agarcharacterized by circular, raised colonies with entire margin, shiny and semi-transparent with a β hemolytic. Morphological examination of G.anatis organisms appeared as Gram-negative, rod-shaped or pleomorphic, cells disterputed singly or in pairs, non-spore forming, non-motile, bipolar staining when stained by giemsa stain. The results of biochemical identification showed characteristic identical biochemical reaction to be G.anatis positive catalase, oxidase, and negative urease and indol test. The phenotypical studies ensured that all isolates were virulent when detected by congo red test. furthermore, the isolates were subjected to PCR for confirmation, where only 4/50 (8%) of isolates were positive for 16S rRNA23S rRNA. As shown in table (2).

Antimicrobial sensitivity test:-
Antibiotic sensitivity test revealed that all isolates were sensitive to gentamicine, ciprofloxacin, cefotaxime and sulfadimethoxine trimethoprim and moderate sensitive to enrofloxacin and amoxicillin claviulinic acid. while, they were resistant to oxytetracyclin and doxycycline.

Molecular characterization of tetH resistant gene of G.anatis:-
The phenotypic resistance of G.anatis to oxytetracyclin and doxycycline could be explained by the presence of (100%) tetH gene in all tested isolates.

Molecular characterization of virulence genes of G.anatis:-
four isolates of G.anatis isolates were examined for detection of ( gtxA (rtxA),gyrB and fifA) genes, the result revealed (100%) detection of gtxA and gyrB genes, while none detection of fifA gene in all tested strains. As shown in table 3.   The molecular investigations of resistance mechanisms of G.anatis isolates to oxytetracyclin and doxycycline which could be explained by the presence of (100%) tetH gene in all tested isolates.
G.anatis infections are usually associated with the presence of virulence genes , G .anatis is able to produce a cytolytic RTX-toxin as a virulence factor which responsible for hemolytic activity against erythrocytes of hosts and leukotoxic activity (Kristensen et al.,2010).In the current study the gtxA gene was detected in all isolates but flfA gene was not detected in all isolates. This result was disagree to that recorded by Sorour et al., (2015) where they detected fifA gene in 50% of isolates. This could be due to the F17-like fimbria is encoded by a four-gene designated as flfD, flfC, flfG and flfA  or due to sub-cultivation of strains which lead to loss of virulence factors expression. .

Conclusion:-
1. G. anatis is one of the most important microorganisms causing salpingitis, peritonitis and drop in egg production. 2. PCR is more accurate than traditional method in detection of G.anatis. 3. RTX-like toxin is the main virulence factor of G. anatis named gtxA which responsible for evoking the phenotypic character of strain such as hemolysis 4. The drug of choice against G.anatis are gentamicin ,ciprofloxacin, cefotaxime and sulfadimethoxine trimethoprim. 5. Further studies and genes sequences required to G.anatis to prepare vaccine against it to overcome the disease and drug resistance.