STUDIES ON CHICKENS ORNITHOBACTERIUM INFECTION AT ISMAILIA PROVINCE

Dept. of Poultry Diseases

Animal Health Research Institute, Ismailia

Studies on chickens Ornithobacterium infection at Ismailia province

(With 6 Tables and 8 Figures)

By

M.K. Moursi and Kawther H. Sabah*

* Dept of Bacteriology, Animal Health Research Institute, Ismailia,

(Received at 22/1/2008)

دراسات على الإصابة بعدوى ميکروب الاورنيثوباکتيريا

فى الدجاج بمحافظة الاسماعيلية

           محمد کمال مرسي دسوقى ، کوثر حسين احمد

أجريت هذه الدراسة لعزل وتصنيف ميکروب الاورنيثوبکتيريا (الأنف والرغام) من دجاج التسمين وأمهات دجاج التسمين في محافظة الاسماعيلية ودراسة مدى استجابتها للمضادات الحيوية المختلفة بالإضافة إلى إجراء العدوى الاصطناعية بالميکروب المعزول. وقد تم  تجميع عدد 115 عينة من دجاج مريض وأخر نافق حديثا من مختلف المزارع التي تعاني من أعراض تنفسية ومصحوبة بتورم في الوجه وذلک من دجاج التسمين وأمهات دجاج التسمين فى أعمار مختلفة. وقد تم إجراء وتسجيل العلامات المرضية الظاهرة على الدجاج وکذلک الصفة التشريحية. ولإجراء الفحص والعزل البکتيرى تم تجميع عينات من (الرئة، القلب، القصبة الهوائية والأکياس الهوائية) من الدجاج. وقد أسفرت النتائج عن أن 27 عينة من أصل 115 کانت ايجابية للعزل لميکروب الاورنيثوبکتيريا وذلک بنسبة 23،47 ٪ وتمثلت فى (16/64 من دجاج التسمين بنسبة 25 ٪  و11/51  من أمهات الدجاج بنسبة 21،56 ٪). وعند إجراء العدوى الاصطناعية لکتاکيت عمر 4 أسابيع وذلک بطرق عدوى مختلفة بالحقن فى الوريد والحقن فى القصبة الهوائية والحقن فى الأکياس الهوائية – أدى ذلک إلى انخفاض في کمية الغذاء وتأخر النمو وتمثل ذلک إحصائيا فى نقص معنوى فى أوزان الطيور المصابة عن الطيور الغير مصابة. وکانت الإعراض عبارة عن انکماش واکتئاب، صعوبة في التنفس، التهاب الملتحمة وتورم العين، العطس وانتفاخ الوجه. وفي حين تفاوتت الآفات التشريحية في شدتها وفقا لطريقة العدوى وعموما کانت عبارة عن التهاب الأکياس الهوائية والقصبة الهوائية ووجود غشاء فبرينى على الکبد والقلب بالإضافة إلى التهابات فى الرئة مع وجود بعض المواد المتجبنة فى الأکياس الهوائية فى بعض الطيور. وسجلت حالتين فقط للنفوق فى العدوى عن طريق الحقن فى الوريد وذلک بمعدل 13.33%. وقد أمکن إعادة عزل الميکروب المحقون من أنسجة الطيور المصابة بشکل تجريبي. واظهر ميکروب الاورنيثوباکتيريا استجابة کانت حساسة لکل من السفتيفيور وکبريتات الکولستين والاريثروميسين والتتراسکلين وبينما کانت المقاومة للجنتاميسن، والانروفلوکساسين والسيبروفلوکساسين وکذلک التراى ميثوبريم.

SUMMARY

A total of 115 diseased and freshly dead broilers and broiler breeders' chickens were collected from different farms at different ages. They were suffering from respiratory signs and facial swelling. Complete clinical and postmortem examinations were recorded. Samples from lung, heart, trachea and air sacs were collected for bacteriological isolation. Antibiotic sensitivity test as well as experimental infection were applied. The results showed that 27 out of 115 (23.47%) were positive for isolation of O. rhinotracheale (16/64 from broilers 25% and 11/51 from broiler breeders 21.56 %). Experimental infection of 4 weeks old chickens via intravenous, intra-tracheal and intra-air-sac leads to decrease in feed intake and growth retardation (The mean of body weight in all infected groups were significantly (P < 0.05) lower than control group, depression, ruffling difficult breathing, conjunctivitis, sneezing, nasal discharge followed by facial edema. Postmortem lesions varied in severity according to inoculated routes and generally were tracheitis, arisaculitis, pneumonia, pericarditis, and perihepatitis, caseated material in air sac of some birds and arthritis. O. rhinotracheale   could be reisiolated from affected organs of experimentally infected birds. Mortality was recorded in two birds only (13.33%) after intravenous inoculation. The isolated O. rhinotracheale were sensitive to amoxicillin, ceftiofur colstin sulphate, erythromycin and tetracycline while were resistant to gentamycin, enrofloxacin, ciprofloxacin and trimethoprim.

Key words: Ornithobacterium O. rhinotracheale, broilers, antibiotic sensitivity

INTRODUCTION

Respiratory infections are still a major problem in poultry and accompanied by heavy economic loss due to increase mortality, increase medication costs, increase condemnation rates, drops in egg production, reduction of egg shell quality and decrease hatchability (Van Epmel and Hafez, 1999).

Since December 1991 respiratory manifestation with different clinical courses have been observed in poultry flocks in different countries (Charlton et al., 1993; Hafez et al., 1993; Hinz et al., 1994; Van Beek et al., 1994). It was initially regarded as pasteurella like organism and finally O. rhinotracheale has been proven to be the primarily pathogen in broiler (Van Veen et al., 2000 a & b). The organism has been isolated from the partridge, pheasant, pigeon, rook, quail duck, ostrich, goose, guinea fowl, chicken and turkey (Charlton et al., 1993; Vandamme et al., 1994 and van Empel et al., 1999).              O. rhinotracheale (ORT)is a Gram-negative, non-motile, pleomorphic, rod-shaped, non-spore forming bacterium. Most strains grow under aerobic, micro-aerophilic, and anaerobic conditions. Optimal growth occurs on 5% sheep blood agar (Van Empel and Hafez, 1999).

Ornithobacterium rhinotracheale infection is an acute highly contagious disease of chickens and turkeys. The severity of clinical signs, duration of the disease and mortality are extremely variable and are influenced by many environmental factors such as poor management, inadequate ventilation, high stocking density, poor litter conditions, poor hygiene, high ammonia level, concurrent diseases and the type of secondary infection (Hafez, 1996).Itis very sensitive to antibiotics and chemical disinfectants (Hafez and Schulze, 1998)but it can acquire resistance against antibiotic easily (Van Emple, 1998), Currently Ornithobacterium rhinotracheale infections may be appeared to become an endemic features, since itcan affect every new restocking even in previously cleaned and disinfected houses especially in areas with intensive poultry production with multiple age farms (Hafez, 1996, Van Empel and Hafez, 1999).

 O. rhinotracheale were able to induce the same kind of respiratory inflammations and weight-gain losses in chickens as well as turkeys after experimental infection Maja et al. (2006a) and Van Empel et al. (1996).

     This study aimed to:

- Isolation and identification of O. rhinotracheale and its incidence in broilers and broilers breeders at Ismailia governorate.

- Experimental infection of isolated bacteria to susceptible chicks via different routes.

- Investigation of antibiotic sensitivity test of isolated bacteria to some    chemotherapeutic agents.

MATERIALS and METHODS

Birds and Samples:

A total No. of 115 (64 broilers and 51 broilers breeders' chickens) were collected from different farms at Ismailia province, they were suffering from respiratory signs and facial swelling. Besides decreased eggs production in broiler breeders. Birds were subjected for clinical and postmortem examinations. Samples from lung, heart, trachea and air sacs were collected for bacteriological isolation.

Bacteriological examination:

Bacterial isolation:

Lungs, heart, trachea and tracheal swabs were inoculated into brain heart infusion broth supplemented with gentamycin as 5-10 µg/ml and incubated at 37°c for 24 - 48 hr., under 5% CO2 tension by using gas bags in candle jar. Then loopfull from cultured broth were streaked on Blood agar with 7% sheep blood either supplemented with gentamycin 10 µg/ml (to inhibit other bacterial growth) or without gentamycin and on MacConkey Agar. The plates were incubated at 37^oC under aerobic conditions as well as at 37^oC under anaerobic  conditions in 5% CO₂ enriched environment by using gas bags in candle jar for     2-3 days according to Vandamme et al. (1994) Traverse et al. (1996) and Rojs et al. (2000).

Identification:

All suspected colonies were subjected for identification by colonial morphology (shape- colour- size- odour) and films were stained by gram stain and conventional biochemical tests were also applied (Van Empel et al., 1997).

Biochemical characterization:

ORT isolates were subjected to standard biochemical tests, including catalase, indole, motility, hydrogen sulfide, carbohydrates fermentations, phenylalanine deaminase, Lysine decarboxylase,              β –Galactosidase, Ornithine decarboxylase methyl red, Voges Proskauer, citrate, urease and gelatine liquefaction, described by (Van Empel et al., 1997).

In vitro sensitivity test:

Determination of in vitro sensitivity pattern of the isolated organism against different chemotherapeutic discs were done. Interpretation of the results of susceptibility findings was done according to a standard protocol for antibiotic sensitivity tests described by the National Committee for Clinical and Laboratory Standards (NCCLS, 2002) and Malik et al. (2003).

Pathogenicity test:

Fifty six, days-old healthy chickens were used and reared at hygienic conditions, food and water were available ad libitum. By the age of 4 weeks; five randomly selected birds were sacrificed and bacteriologically tested before experiment to prove that they were free from ORT. The other birds (60) were divided into 4 groups each of 15 and housed separate as follow:

Group 1: chickens were inoculated intravenously with 1ml of a whole culture of brain heart infusion broth adjusted to account of approximately 1 X 10⁷ colony forming units/ml according to Saeb et al. (2002).

Group 2: chickens were inoculated intra-air-sac with 1ml of inoculum of ORT according to Saeb et al. (2002).

Group 3: chickens were inoculated intratracheally with 1ml of a whole culture of brain heart infusion broth adjusted to account of approximately 1 X 10⁷ colony forming units/ml according to Saeb et al. (2002).

Group 4: chickens non-inoculated and served as control.

Parameters of infection: During 7 to 14 days after O. rhinotracheale challenge, morbidity and mortality were recorded, the daily weight gain after challenge was determined, macroscopical lesions were recorded at postmortem examination of the birds, and attempts to reisolate the challenge bacteria were carried out. At postmortem examination, a scoring system for the observed lesions was used according to (Van Veen et al.,2000 a & b)as follows:

For air sacs:

 0 = no abnormalities

 1 = one air sac seriously affected by fibrinous airsacculitis or limited  pin-head sized foci of fibrinous exudate in both air sacs

 2 = both air sacs seriously affected by fibrinous airsacculitis;

For lungs:

0 = no abnormalities

 1 = unilateral pneumonia

 2 = bilateral pneumonia.

 For trachea:

0 = no abnormalities.

 1 = some exudates in the tracheal lumen

 2 = lumen of the trachea filled with exudates.

Statistical analysis: The statistical analyses for the weights and weight gains were performed by using Student’s t-test (Snedecor & Cochran, 1967).

RESULTS

Clinically affected bird showed depression, roughled feathers, decreased feed intake, reduce weight gain, sneezing, nasal discharge and facial edema. while the postmortem examination revealed uni- or bilateral consolidation of the Lungs fibrinous pericaditis airsacullitis tracheitis in some cases swelling of the liver and spleen

Bacterial examination:

Small pin­point grey colonies, sometimes with a reddish glow, non hemolytic varied in diameter were observed after 24 or 48 hr of incu­bation on blood agar. Growth was observed un­der both aerobic or microaerophilic conditions. No growth on MacConkey agar was seen. All isolates were gram negative, pleomorphic, rod-shaped bacteria.

            Biochemical and enzymat­ic results obtained with routine laboratory re­agents were uniform among isolates, showing oxidase positive, ferments glucose, lactose, galactose and fructose but doses not ferment sucrose and maltose. It was negative for catalase, methyl red, voges proskauer, gelatin liquefaction, citrate utilization, indole, nitrate reduction and triple sugar iron (Table 2). According to the cellular and colonial morphology of the organism and based on biochemical and enzymatic characteristics, by laboratory procedures, all isolates were identified as O. rhinotracheale.

        The incidence of O. rhinotracheale isolation was 23.47% from the total examined samples of (lung, trachea, airsac). 27 out of 115 were positive for isolation of O. rhinotracheale (16/64 from broilers 25% and 11/51 from broiler breeders 21.56 %) as shown in Table (1). Table (3) showed the results of antibiotic sensitivity test of isolated                      O. rhinotracheale.

Experimental studies:

The most prominent clinical signs 1^st & 2^nd weeks after experimental infection appeared to be decreased feed intake and growth retardation (The mean of body weight in all infected groups were significantly (P < 0.05) lower than control group) Table (6), depression, ruffling difficult breathing, mild conjunctivitis, sneezing, nasal discharge lacrimation followed by facial edema (Fig. 1), unilateral swelling of infraorbital sinuses (Fig. 2).

Post-mortem lesions were tracheitis, uni or bilateral pneumonia  (Fig. 3), airsacculitis, slightly congested and enlarged liver & spleen, fibrinous airsacculitis, tracheal exudates, fibrinous percarditis & perhepatitis (Fig. 4), accumulation of fibrin and caseated materials in abdominal cavity (Fig. 5), patchy areas of fibrin on lungs surface. Some variations in signs and postmortem lesions were found between the inoculated routes (Table 4 & 5). Moreover I/V inoculation resulted in airsaculitis, pneumonia and severe arthritis in some birds and the bird could not stand (Fig. 6). The inoculated bacteria could be reisolated at day 7 postinoculation from the heart, liver, spleen, air sacs, and lung (Table  4).

The in­tratracheal challenge resulted in airsaccu­litis, hydropericardium, pneumonia and mild arthritis. The inoculated bacteria could be reisolated from the tra­chea, lung, and air sac of inoculated chick­ens (Table 5).

            The intra-air sacs challenge resulted in severe airsacculitis, accumulation of fibrin in abdominal cavity, pericarditis, perihepatitis, liver and heart adhesion. Bacteria were reisolated from the heart, lung, and air sac (Table 5). The mortality rate was recorded only in two birds (13.33%) after I/V inoculation. No deaths were observed in other groups.   

Table 1: Incidence of O. rhinotracheale isolation from different examined flocks.

Table 2: Biochemical prosperities of isolated O. rhinotracheale

Table 3: In vito sensitivity test of O. rhinotracheale

Table 4: The pathological score lesion of experimentally infected chicks with O. rhinotracheale

Table 5: The postmortem lesions observed 2wk post-experimental infection via three inoculation routes with O. rhinotracheale (ORT) and their reisolation.

A/S = airsacculitis; P = pneumonia; Ar = arthritis, swollen joint with caseous material;                         HP = hydropericardium; PH/PC = fibrinous perihepatitis and pericarditis with adhesions.

Table 6: The effect of O. rhinotracheale infection on average body weight and average daily weight gain.

Within columns, averages having different letter are significantly different (P<0.05)

List of figures

Fig. 1: Conjunctivitis, lacrimation and facial edema of experimentally infected birds.

Fig. 2: experimentally infected chicks showed swelling of the infraorbital sinus accompanied by swollen head.

Fig. 3: Lung pneumonia in 4 weeks old broiler showing clear boundary between affected and non affected parts of the lung.

Fig. 4: Fibrinous pericarditis, perihepatitis and heart adhesion in experimentally infected chicks.

Fig. 5: Airsacculitis of abdominal air sac with accumulation of yellow caseous materials in abdominal cavity.

Fig. 6: Airsacculitis of thorasic air sac with large clots of fibrin experimentally infected chicks.

Fig. 7: Experimentally infected chicks showed legs affection, apart away and could not stand.

Fig. 8: Heart experimentally infected chicks showing fibrionus pericarditis.

DISCUSSION

Respiratory disease conditions due to bacterial and viral agents are continuing to cause heavy economic losses in the poultry industry worldwide. Ornithobacterium rhinotracheale has been recognized in many countries worldwide and incriminated as a possible additional causative agent in respiratory disease complex. (Hafez, 2002).

In this study the clinical signs of naturally affected birds weredepression, roughled feather, decreased feed intake, reduced weight gain, sneezing, nasal discharge and facial edema while the gross lesions in general weresevere pneumonia, airsaculitis, tracheitis, oedema, uni- or bilateral consolidation of the lungs with fibropurulent exudates, pericarditis, airsacculitis, peritonitis.These findings are similar to those reported byHafez, (2002) and Soriano et al. (2002)  

Bacteriological examination revealed that the isolation of Small pin­point grey colonies, sometimes with a reddish glow, non hemolytic varied in diameter were observed after 24 or 48 hr of incu­bation on blood agar. All isolates were gram negative, pleomorphic, rod-shaped bacteria showing oxidase positive, ferments glucose, lactose, galactose and fructose but doses not ferment sucrose and maltose and for other tests it was negative for catalase, methyl red, voges proskauer, gelatin liquefaction, citrate utilization, indole, nitrate reduction and triple sugar iron.

All isolates could be identified as O. rhino­tracheale by bacteriological labo­ratory tests. This result was completely agreed with biochemical and en­zymatic properties of O. rhino­tracheale reported by Charlton et al. (1993), Van Empel et al. (1997) Soriano et al. (2002) and Canal et al. (2005).

Ornithobacterium rhinotracheale was isolated from lung, air sac and trachea of examined birds as that reported by Saeb et al. (2002) andSoriano et al. (2002).

The incidence of O. rhino­tracheale isolation in this study was 23.47% (27/115) from total examined samples (16/64 from broilers, 25% and 11/51 from broiler breeders, 21.56 %) this result is much less than that reported by Abden and lotfy (2006) they reported that              O. rhinotracheale was isolated by 75% of total examined chicks while, Shahata et al. (2006) isolated O. rhinotracheale at 34% from lung, trachea and air sac of examined chickens and layers. In contrast Shihata and Ibrahium (2004) isolated O. rhinotracheale by 11.72%. while Saeb et al. (2002) recoverd O. rhino­tracheale by 8.8%. Also Türkyilmaz, (2005) isolated O. rhinotracheale from broiler breeders of 37, 42 and 46 week old by 1.2%.

Since a standard protocol for antibiotic sensitivity tests for        O. rhinotracheale does not exist we depended upon the method described by the Clinical and Laboratory Standards Institute (CLSI, 2002) for fastidious Gram-negative organisms.

With regard to antibiotic sensitivity testing, the isolated             O. rhinotracheale were sensitive to amoxicillin, ceftiofur colstin sulphate, erythromycin and tetracycline while were resistant to gentamycin, enrofloxacin ciprofloxacin and trimethoprim. This results are in agreement with these reported by Malik et al. (2003), Türkyilmaz, (2005) and Maja et al. (2007). Maja et al. (2006 a) reported that Acquired fluoroquinolone resistance is commonly encountered in ORT isolates. On the other hand, Shahata et al. (2006) found that amoxicillin, tetracycline and enrofloxacin were the most effective drugs against isolated ORT in vitro. Moreover, Saeb et al. 2002, found that                O. rhinotracheale isolates were sensitive to tetracycline and to lesser extent to other antibiotics. Such differences in the antibiotic susceptibility pattern may be due to misuse and /or frequent exposure of the bacteria to antibiotics in field.

The results of experimental infec­tions of O. rhinotracheale revealed that the three inoculated routes resulted in growth retardation, depression, ruffling, difficult breathing, conjunctivitis, sneezing, nasal discharge, lacrimation followed by facial edema, unilateral swelling of infraorbital sinuses. These results are in agreement with the  studies of Travers et al. (1996) and Saeb et al. (2002) .Van Empel et al. (1996) reported that the most prominent clinical signs after experimental infection appeared to be the growth retardation, dyspnoea, mucus discharge and mortality.

In this study mortality was recorded in only two birds (13.33%) after an intravenous injection with O. rhinotracheale.Goovaerts et al. (1998)found that only an intravenous challenge was able to induce 20% mortality in experiemntallyO. rhinotracheale chicks,whileShahata et al. (2006) observed that the mortality was 30% in experimentally infected birds. Also Shihata and Ibraheem (2004) found that moralities were 10% and 20% in hubbard and balady chicks respectively after experimental infection.

The result of gross lesion revealed development of different records of post­mortem findings post-experimental infection via three inoculation routes. These findings were airsacculitis, pneumonia and peri­carditis perihepatitis, and liver and heart adhesion and joint affection which are in agreement with the results obtained when similar inoculation routes were applied by Saeb et al. (2002) and Shahata et al. (2006). Also Ryll et al. (1997a); Sprenger et al. (1998)reported that airsacculitis, pneumonia and increased mortality were observed after aerosol, intra-tracheal, intravenous and/or intra-thoracic infection.

            Neverthe­less, not all of the clinical signs and/or post­mortem findings that were observed in the nat­ural outbreak could be reproduced experimen­tally. This discrepancy between natural and experimental      O. rhino­tracheale infection might be explained by differences in aggravating and predisposing factors such as stress, high stock density, poor ventilation, presence of other bacteria, or high ammonia levels in field conditions.

The obtained result in this study indicated obviously variations in pathogenicity of O. rhinotracheale among the inoculated routes. Similar conclusions were reported by other researchers (Van Empel et al., 1996 and Travers et al., 1996)they found that the severity of clinical signs and lesions could be referred to route of infection and variations in the virulence and pathogenicity of O. rhinotracheale.

In conclusion, our results indicate that O. rhi­notracheale was isolated by 23.47% from examined cases of broiler and broiler breeders at Ismailia province. Challenge test suggests that this bacterium was able to produce respi­ratory signs with various postmortem lesions in experimentally infected chicks and considered as a primary pathogen for chickens.  

Acknowledgment

Authors would like to thanks Dr. Amin A.A. Assis. Prof. Animal Breeding and Genetics Dept. of Animal Prod. Fac. of Agri. Suez Canal Univ. Ismailia Egypt, for his statistical analysis helps in the present study.

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