Animal Health Research Institute, Ismailia

Epidemiological studies on bacteriological aspects of air sacculitis in chickens

(With 5 Tables and 4 Figures)

By

Fatma M. Yousseff; Mona, A. Ahmed

 and Dalia H. Mansour*

* Dept. of Poultry & Rabbit Med., Faculty of Vet. Med.,

Suez CanalUniversity.

(Received at 15/6/2008)

دراسات وبائية على المسببات البکتيرية لالتهاب الأکياس الهوائية

في الدواجن

فاطمة محمد احمد يوسف ، منى عبدا للاه احمد ،  داليا حامد منصور

أجريت هذه الدراسة الوبائية على خمس مزارع دواجن بمحافظة الإسماعيلية وکانت الاعراض المميزة أعراض تنفسية مصحوبة بالتهابات معوية. کما أسفر التشريح المرضي للطيور المصابة عن وجود  صورة من التسمم الدموي. وبإجراء الفحص البکتيري تم عزل الميکروب القولوني (60 & 40%)، بينما عزل ميکروب الاورنيسو بنسبة (26.6 & 20%) من کتاکيت التسمين والدجاج البياض على التوالي. کما أسفرت نتائج تصنيف الميکروب القولوني إلى  85 عتره وکانت کالأتي : سبعون صنفوا إلى 6 أنواع مختلفة و15 عتره غير محدده. وکل العترات المعزولة من الميکروب القولوني قد تم تقييم مدى ضراوتها باستخدام صبغة الکونجو والنشاط التحليلي للدم والقدرة على حدوث التسمم. کما تم استخدام المضادات البکتيرية على الميکروبات المعزولة. وقد أجريت  العدوى التجريبية لعتره الاورنيسو التي تم فحصها وتسجيل نتائجها أسبوعيا". وقد أسفر العلاج في التجربة بالانروفلوکساسين عن نجاحه وحدوث الشفاء من الأعراض التنفسية. ومن تلک النتائج يمکن استنتاج أن التهاب الأکياس الهوائية يکون منتشرا في کتاکيت التسمين والدجاج البياض بمحافظة الإسماعيلية. وان الميکروب القولوني هو الميکروب الأول في حدوث المرض ويليه ميکروب الاورنيسو. لذلک يجب إتباع الأسلوب الأمثل في النظافة لتقليل عدد الميکروبات المحيطة بالطيور.

SUMMARY

This study was carried out on five poultry farms at Ismailia Governorate suffering from respiratory symptoms accompanied with gastroenteric findings. Post mortem findings revealed septicemia. Bacteriological examination revealed the isolation of E. coli (60 %& 40%) while ORT was (26.6% & 20%) and E. coli concomitant with ORT were (18.6% & 12%) in broilers and layer respectively. The eighty-five E. coli isolates were as follow: seventy were identified to six different serotypes, O78, O26, O1, O2, O157 and O111, with different frequencies and fifteen untypable. All E. coli isolates tested for evaluation of it's virulence by Congo red binding assay, Hemolytic activity, Invasiveness assay and Enterotoxins assays. The in vitro Antimicrobial resistance patterns of the isolates were reported. Experimental infections of O. rhinotracheale  were examined and scored weekly. Recovery from respiratory disease was overall most successful after Enrofloxacin treatment. It could be concluded that air sacculitis is prevalent among broiler and layers chickens at Ismailia province. E. coli is the most commonly incriminated agent and Ornithobacterium rhinotracheale stands as the second most important pathogen. Therefore, management and sanitation practices designed to reduce the number of these types of organisms in the birds' environment are necessary.

Key words: Poultry, E.coli, Ornithobacterium rhinotracheale,

 and Air sacculitis

Introduction

"Air sacculitis" means any infection or inflammation of the bird air sacs, which mainly appear in chronic cases. Poor blood supply to the air sacs makes removal of inhaled microorganisms very difficult by the bird's immune system. Signs of air sacculitis may be present at rest or only after flying, coughing, wheezing, and labored respiration (Pesek, 2000). Air sacculitis is a leading mortality and a significant cause of carcass condemnation in broilers. Air sacculitis is a respiratory disease of poultry, frequently caused by E. coli. However, outbreaks of air sacculitis caused by E.coli acting as a primary pathogen have been documented (Stebbins et al., 1992). It is characterized by thickened, inflamed air sacs with fibrinous exudates, pericarditis and perihepatitis as sequalae of a coli- septicemia (Gross, 1994). Biological and environmental stresses such as viral infection, overcrowding, and poor ventilation predisposebirds to E. coli infections (Mellata et al., 2003).Escherichia coli causes a variety of diseases in poultry, including respiratory tract infection, Omphalitis, swollen-head syndrome, enteritis, septicemia, and cellulites (Gross, 1994 and Norton, R.A. 1997) and these diseases are responsible for major economic losses in the chicken industry.

Ornithobacterium rhinotracheale (ORT), which is a respiratory pathogen, has been described by Vandamme et al. (1994). It was initially regarded as a Pasteurella-like organism. ORT can cause severe respiratory clinical signs in turkeys and chickens (Hinz et al., 1994 and Sprenger et al., 1998). ORT was proven to be a primary pathogen in broilers (Van Veen et al., 2000) and potentially pathogenic for fowl (Sprenger et al., 1998; Van Empel et al., 1999 and McMullin, 2004). Although ORT infections are frequently associated with other respiratory diseases, ORT has sometimes been isolated from asymptomatic flocks (Charlton et al., 1993 and McMullin, 2004). ORT is often overgrown by E. coli, Proteus spp. and Pseudomonas spp., which have a rapid growth rate, especially in materials taken from hens (Türkyilmaz, 2005). Blood-stained mucus in the mouth is an occasional finding. The organism has been isolated from the partridge, pheasant, pigeon, rook, quail duck, ostrich, goose, guinea fowl, chicken and turkey (Vandamme et al., 1994; Van Empel et al., 1999; Charlton et al., 1993 and McMullin, 2004). The disease has been described in many countries (Charlton et al., 1993 and Erganifl et al., 2002 and McMullin, 2004). In Turkey, ORT isolations were first obtained from two commercial pullets aged 12 and 15 weeks (Türkyilmaz, 2005). The most common post mortem lesions were pneumonic lungs, pleuritis with accumulation of creamy "yoghurt-like" exudates (Hafez, 2002).         

The aim of this work to investigate the bacterial causes of air sacculitis in broiler and layer at Ismailia province and their associated with antimicrobial susceptibility in affected chickens and application of  antibiotic of choice. 

MATERIALS and METHODS

 1-History and clinical examination:

A total number of 125 chickens (moribund and freshly dead) were clinically examined in this study. Broiler samples (75 broilers) were collected from three broiler chicken farms (aged from 17 to 35 days). Layer samples (50 layers) from two layers farms (aged from 18-24 months). The collected samples were mainly from birds suffering from respiratory signs and swollen head accompanied with gastroenteric findings and cyanosed comb and wattles.

2-Samples:

Tracheal swabs, lungs and air sacs, liver, and heart blood were collected from 75 broilers, and 50 layers for bacteriological examination

3-Bacteriological examination:      

3.1- Isolation of E. coli: was carried out according to Quinn, et al. (2002). Where the serological typing of the E. coli isolates was carried according to Edwards and Ewing (1972).

3.2- Isolation of ORT: Tracheal swabs were aseptically inoculated into brain heart infusion broth supplemented with 10 µg/ml Gentamycin according to Back et al. (1997) and incubated at 37°C for 24 -48 hours., under 7.5–10% CO2 tension by using gas bags in candle jar according to Vandamme et al. (1994); Traverse et al. (1996) and Zorman-Rojs et al. (2000). Then that loop full from the cultured broth was streaked onto blood agar supplemented with 7% sheep blood and 10 µg/ml gentamycin (to inhibit growth of other bacteria). The plates were incubated in a 7.5–10% CO2 atmosphere at 37°C for at least 48 hours. Storage of isolates was according to Chin et al. (2003).

3.3- E. coli serotypes were tested for evaluation of its virulence by application of:

A-Congo red binding assay (Berkhoff and Vinal, 1986). Tested strains were examined for its growth on Congo red medium. The reaction appeared after 24 hrs of incubation at 25°C and left for 4 days the CR positive was in-dicated by the development of bright or orange red colonies, and the negate-ve isolate appeared white colonies.                                                               

B- Hemolytic activity(hemolysin assays)according toBeutin et al. (1989).

 C-Invasiveness assayaccording toFeingold and Baron (1986):The ability of E. coli strain to invade epithelial cell were tested in rabbit eye model "Sereny Test". Cultures were grown onto nutrient agar plates and were washed off in phosphate buffer saline to give a suspension of approximately 10⁸ cfu/ml. A single drop from the suspension was allowed to fall into the eye of a rabbit while the eye lid was held a part. The other eye was dropped by phosphate buffer saline and kept as a control. The invasive organism was allowed to penetrate the epithelial cells, causing a purulent and oxidative conjunctivitis and compared with the other normal eye.

D- Enterotoxins assays: The ability of the isolated strains of E. coli to produce enterotoxins was assayed by the infant mouse testaccording toErganis et al., (1986). A volume of 0.1 ml of culture filtrates of E. coli.Isolates was injected through the abdominal walls into the milk-filled stomach of infant mice, which were 2 to 4 days old. Also, there were two infant mice injected by 0.1 ml of saline and were used as negative control. After 4 hours, the mice were sacrificed and the entire intestine was removed. The intestine and the remaining body were weighed to calculate the ratio of intestine weight/ remaining body weight. A ratio greater than 0.085 was recorded as positive test forenterotoxins.

4- Antimicrobial susceptibility tests: The Bauer–Kirby technique (Bauer et al., 1966)was adopted. Muller–Hinton agar (Oxoid, Basingstoke, UK) was prepared in a uniform thickness (4 mm) for testing of E. coli isolates, whereas 5% sheep blood agar was prepared for ORT. The following anti-microbial agents (ARCOMEX, co.), which represent the commonly used antibiotics, were tested: Enrofloxacin (5 µg), Ofloxacine (5µg), Amikacine (30µg), Neomycin (30µg), Flumequine (30µg), Amoxicillin (10), Tetracycline (30 µg), Gentamycin (10µg), Oxytetracycline (30 µg), Danofloxacin (5µg),  Sulfamethoxazole-trimethoprim (5 µg), Cholormphenicol (30 µg), and Ampicillin (10 µg).

5- Pathogenicity test of O. rhinotracheale. Forty five healthy Cobb chickens 21 days old were used for that trails.  Five birds from them were tested before experiment and proved free from ORT organism. The other birds were equally divided into four groups: The 1^st group challenged by aerosol with 1ml of whole culture of brain heart infusion count of approximately 10⁸ c.f.u/ml according to El-Sukhon et al.(2002). The second and the third   groups were inoculated with aerosol route and treated with Amoxicillin, Enrofloxacin. The fourth group was inoculated with saline and kept as negative control. Drugs in the second and the third groups were applied into drinking water for five successive days. Chickens kept under observation for 3 weeks post infection for clinical signs and mortality. Two chicks were sacrificed at 7, 14 and 21 days post infection and all dead chicks were examined for score lesions.   Re-isolation of ORT from experimentally infected birds was done. Score lesions post infection was done according to Van Veen et al., (2000). As follow:  Lung = 0 (no abnormalities), 1=unilateral pneumonia, 2=bilateral pneumonia. for Trachea=0 (no abnormalities), 1= Exudates in trachea, 2=trachea filled with exudates, Air sacs 1= fibrinous exudates

6- Therapeutic drugs: Enrofloxacin (Baytrail®10%) Bayer 10mg/kg body weight. Amoxicillin 0.1% (Pfizer) were added in drinking water.

RESULTS

The clinical signs and post mortem findings: among the affected birds varied according to the severity of the disease. Facial edema, conjunctivitis, swollen sinuses beside closed eyes, diarrhea, conjunctivitis and cyanosed comb and wattles were the most common signs. Other signs as emaciation, dullness, inappitance and recumbence before death were recorded.  Moreover, some respiratory signs in the form of sneezing, coughing and gasping were observed in some birds.  Postmortem findings from naturally infected dead chicks or layers revealed air sacculitis, pneumonia, tracheitis, edema in face and sinus and the head appear cyanotic, inflammation of oviduct and a picture of septicemia, congestion of the liver and lung with enlargement of the heart and thick pericardium were seen.  Morbidity in farms ranged from 4-7% and mortality ranged from 5-12% (Table 1).                                                             

Bacteriological examination showed that 65 (45 broiler chicks and 20 layer) E. coli isolates from air sacculitis cases.  30   strains were identified as O. rhinotracheale (10 broiler chicks and 20 layers). 20 samples (14 broiler chicks and 6 layers) were showed to be mixed E. coli with O. rhinotracheale (Table 2).  Seventy from the 85 E. coli isolates were identified and belonged to 6 different serotypes, namely O78,O26, O1,O2, O157and O111, with frequencies of 24, 16, 12, 10, 5and 3 respectively (Table 3). However, the remaining 15 E. coli isolates were untypable.                                                                                           

Biological characteristics and virulence factors of E. coli isolated from diseased, dead and slaughtered birds were tabulated in Table (3).           Enterotoxins assays  revealed that two E. coli strains recovered from birds were entero-toxigenic and caused accumulation of fluid in the intestinal tract of infant mice belonged to serotype O26 (16/70) 22.86% and O157 (4/70) 5.71%. The results of antibiogram for isolated microorganisms recovered from diseases, dead and slaughter birds were illustrated in Table (4).

Pathogenicity test of O. rhinotracheale: The results of clinical signs in experimental infection with ORT non treated group revealed the same signs as natural infection, figure (1, 2, 3, and 4) during the observation period (21 days). Mortality was 20% (2/10) and significant decrease in body weight after 14 days of infection. While in-group 2 (infected and treated with Amoxicillin) the mortality was 10% (1/10) without significant decrease in body weight, while the third treated group with Enrofloxacin (Baytrail®10%) showed improvement in health condition, no mortality and non significant decrease in body weight. The results of postmortem lesion were similar to the natural infection (air sacculitis, pneumonia, pericarditis and liver congestion). The re-isolation trials were positive from trachea, air sacs, and lungs from the infected non treated group.

DISCUSSION

Respiratory diseases are one of the most common disease problems affecting poultry world wide. Respiratory diseases result in serious economic losses to the poultry industry worldwide (Rosenberger, 1985). Various bacterial and viral pathogens are encountered in respiratory disease in domestic poultry (Chin and Droual 1997 and El-Sukhon et al. 2002). Colibacillosis is a respiratory diseases causing major problems, which face poultry industry. It is responsible for drastic chicken morbidity and mortality in Egypt.

Ornithobacterium rhinotracheale has been isolated in 1994 by Vandamme et al. (1994) and named ORT. It has recognized in many countries and is considered an important agent in respiratory diseases in chickens and turkeys ( Joubert et al., 1999). In Egypt, El Gohary and Awaad (1998) found that ORT has been incriminated as possible causative agent in respiratory diseases complex either alone or in synergy with other infectious or non infectious agent.

  Table (2) showed the incidence of O. rhinotracheale from broiler chickens and layers suffering from airsacculitis was (26.6%), and (20%), respectively. ORT isolation in association with the isolation of E. coli represent 18.6 % and 12% respectively. Our results agreed with such an association of O. rhinotracheale with E. coli was reported by Sakai et al. (2000) and El-Sukhon et al. (2002).

Isolation of E. coliin a much higher percentage (60% & 40%), more than that of O. rhinotracheale (26.6% & 20%), because E. coli is common in the upper respiratory tract of chickens, and the stress factors may facilitate its spread with possible subsequent air sacculitis  (Rosenberger et al., 1985). The obtained results were consistent with the results recorded in some other parts of the world (Lambie et al 2000, Sakai et al., 2000 and El-Sukhon et al., 2002). Different serotypes of E. coli associated with respiratory infection in chickens were reported by Allan et al. (1993) and Chin et al. (2003). In this study, serotype O78 was the most common (28.2%) followed by serotypes O26 (18.8%), O1 (14.11%), O2(11.76%), O157 (5.88%). and O111 (3.5%). These results agreed to some extent, with some other studies (Al-Tarazi, 1983; Allan et al., 1993 and Chin and Droual, 1997).

Isolated strains of  E. coli , which recovered from diseased birds, could bind Congo red (100%), thus the use of Congo red dye was proposed with the objective of distinguishing between pathogenic and non-pathogenic microorganism, our results agreed with Berkhoff and Vinal (1986); Nagwa et al. (2001) and Amany and Ebtehal (2002).  Pathogenic  E. coli have some products associated with virulence of organism, they include b-hemolysin which was observed in 48 out of 85 isolates (56.47%). our results agreed with Holland et al., (1996) and Amany and Ebtehal (2002) which used b-hemolysin producer as a virulence factor.

Our results of antibiogram in vitro showed 100% the E. coliisolates were resistant to Amoxicillin, Tetracycline, Oxytetracycline, Danofloxacin and Ampicillin. Moderate sensitive to Gentamycin and Flumequine but meanwhile, high sensitive to Enrofloxacin, Amikacine and Ofloxacine. The obtained results agree with other reported results by Lambie et al. (2000); El-Sukhon et al. (2002) and Marien et al. (2007).  Enrofloxacin is frequently, used in the treatment of E. coli infection in poultry (Gross 1984). ORT isolates were highly sensitive to Enrofloxacin, Tetracycline, and Amoxicillin moderate to Ampicillin, Neomycin, and Streptomycin and as resistance to Flumequine, Amikacine and Gentamycin. These results agree totally or partially, with some reports (Abdul-Aziz and Weber 1999 and Zorman-Rojs et al., 2000) Also these result agreement with Marien et al. (2007) who reported the successful Enrofloxacin effect on ORT and Shahata et al. (2006) who reported the ORT sensitivity to Flumequine and Gentamycin. On the other hand these results disagreed with Türkyilmaz (2005) how reported that the ORT resistance to Enrofloxacin.

Ornithobacterium rhinotracheale was determined, in broiler and layer. Similar results were reported in different parts of the world (Van Empel et al., 1997; Joubert et al., 1999; Sakai et al., 2000 and El-Sukhon et al., 2002). The natural infection with ORT was accompanied by decreased water and feed in take (Van Empel et al., 1999), dyspnea, mucous discharge, and high mortality (Sakai  et al., 2000; Sprenger  et al., 2000 and El-Sukhon et al., 2002).  

The experimental infections of ORT revealed growth retardation, cyanosed head, facial edema, conjunctivitis beside closed eyes (Figure 1, 2) and mortality which recorded in two bird after an aerosol inoculation with ORT isolate. These results were in agreement with other studies  recorded by Van Empel et al. (1996). The Mortality and lesion score tabulated in Table (5) were 20%, 10% and 0% in the infected none treated, treated with Amoxicillin, and treated with Enrofloxacin groups respectively. This results is similar to that reported by Goovaerts et al. (1998) and Shahata et al. (2006). Postmortem findings were (air sacculitis, pneumonia and pericarditis, and liver necrosis) as shown in figure (3, 4) were in agreement with other results obtained by Van Veen et al. (2000). Nevertheless, not all of the clinical signs and/or postmortem findings that were observed in the natural outbreak could be recorded experimentally (Van Empel et al., 1996), possibly because of some environmental field stress conditions or infection with other pathogens (Zorman-Rojs et al., 2000). Treatment trails based on the results of in vitro antibiogram. Treated group with Enrofloxacin was superior over non treated group and followed with Amoxicillin treated group.

We conclude that air sacculitis in Ismailia province is common among broiler chickens and layers. Escherichia coliis the most commonly incriminated agent and its serotypes O78, O26, O1, O2, O157 and O111 were the most commonly encountered serotypes. Ornithobacterium rhinotrachealestands as the second most important pathogen. So, management and sanitation practices designed to reduce the number of these types of organisms in the birds' environment are necessary. In addition, reducing stress factors and other disease agents can enhance the ability of birds to defend against harmful infections.

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Table 1: Bird's morbidity %, and mortality % from affected flocks in Ismailia province.

Table 2: Incidence of E.coli and ORT from different organs from both broiler and layer flocks.

                             Table 3: Biological characteristics of E.coli (85 isolates) sero-groups isolated from diseased, dead

                                             and slaughter birds.

   No. =positive number   percentage was calculated according to number of examined E.coli