STUDY ON PROTEOLYTIC BACTERIA AFFECTING THE RESPIRATORY TRACT OF CHIKENS دراسة عن البکتريا المحللة للبروتيـن المؤثرة على الجهاز التنفسي للدجاج

STUDY ON PROTEOLYTIC BACTERIA AFFECTING

THE RESPIRATORY TRACT OF CHIKENS

Hebat Allah A.E Mohamed^*, H. Kh Hassan^* and T. Y. Abd El-Motelib^**

*Animal Health Research institute, Assiut Provincial laboratory

**Dept. of poultry Diseases, Faculty of Veterinary Medicine, Assiut University

INTRODUCTION:

Easterday and Hinshaw, (1991) reported that bacteria in the respiratory flora of poultry secretes proteolytic enzymes and so are named proteolytic bacteria. These enzymes may increase the severity of avian influenza virus in poultry due to cleavage of avian influenza virus hemagglutinin molecules by proteolytic enzymes. The potential existence of this mechanism is especially appealing as an explanation for the frequent observation that many A1 viruses associated with severe clinical illness under field conditions are non pathogenic under laboratory conditions (Newman et al., 1981), and (king et al., 2009).

Byrum and Slemons (1995) showed that proteolytic bacteria were readily demonstrated among the upper respiratory tract flora of poultry included five species of Staphylococci (Staph. aureus, Staph. epidermis, Staph. sciuri, Staph. xylosus and Staph. hyicus) as well as two gram–negative bacterial species: Flavobacterium sp. and Vibrio alginolyticus, where the later 2 organisms usually reported to be associated with soil and surface water origins. The genus Vibrio found in fresh water, brackish or estuarine water and marine or salt water. The large number of proteolytic bacteria in the marine environment, coupled with the multiplicity of enzyme production by each bacterium, (Merkel 1971). Mancini et al., (2008) reported that interaction of extracellular proteases with the hemagglutinin may activate viral infection in natural infection.

The present study was under taken to cover the following points:

-Isolation and identification of proteolytic bacteria which affect the respiratory tract.

- Experimental infection of chicks with isolated proteolytic bacteria.

- In vitro sensitivity test to show the most effective drugs against the isolated organisms.

MATERIAL AND METHODS:

Material:

Specimens:

Ninety five samples (trachea) from alive and freshly dead chickens (different ages) with respiratory signs were collected from different farms of Assiut Governorate.

Media, sugars used were:

Tryptose soy agar with 5% sheep blood, caseinate agar, MacConkey's agar sodium chloride, glucose, mannose, sucrose, maltose, mannitol, lactose, esculin, yeast extract, ferric citrate, urea agar and gelatin.

Reagents:

Kovac's, oxidase and Voges – Proskauer :

Stain : Gram's stain.

Chicks for experimental infection :

Fifty, 1-day-old chicks were kept under observation for one week before the experiment.

In vitro antibiotic sensitivity discs used were:

Neomycin (30 μg), gentamycin (10 μg), ampicillin (30 μg), amikacin (30 μg), tetracycline (30 μg), spectinomycin (25 μg), streptomycin (10 μg), amoxycillin (25 μg), penicillin (10 μg), enrofloxacin (5μg), kanamycin (30 μg), cephalexin (30 μg), oxytetracycline (30 μg) and doxycycline (30 μg).

Methods:

Isolation:

Tracheal samples were inoculated on to caseinate agar and tryptose soy agar with 5% sheep blood. The bacterial culture plates were incubated aerobically at 37^oC for 24-48h. Susbected colonies were subjected to bacteriological examination to identify the organism by showing: (shape – size - colour) of the colonies, the typical morphology of the organisms by Gram's stain and studying the biochemical reactions according to Ellen et al., (1994) and Connie and George (1995).

Pathogenicity test:

Fifty, 1-day old balady chicks were used. They kept under observation for 7 days before the experiment, then they were divided as follow:

1^st group: Ten, 1-week-old chicks were inoculated intranasally with 0.5 ml of a bacterial suspension of the isolated Staph. hyicus containing 10⁶ C.FU of 24 h. culture of blood agar.

2^nd group: Ten, 1-week-old chicks. were inoculated orally with 0.5 ml of a bacterial suspension of the isolated Staph. hyicus containing 10⁶ CFU/ml of 24h. cultures of blood agar.

3^rd group: Five, 1-week-old chicks were left as control .

4^th group: Ten, 1-week-old chicks, were inoculated intranasaly with 0.5 ml of a bacterial suspension of the isolated Flavobacterium sp. containing 10⁶ CFU/ml of 24 h. culture of blood agar.

5^th group: Ten, 1-week-old chicks, were inoculated intranasally with 1ml of a bacterial suspension of the isolated Flavobacterium sp. containing 10⁶ CFU/ml of 24 h. culture of blood agar .

6^th group: Five -1week-old chicks were left as control.

In vitro antibiotic sensitivity test:

Sensitivity test were carried out on blood agar using sensitivity discs.

RESULTS:

Postmortem (PM) examination of naturally infected chickens reveald accumulation of mucus or bloody mucus in the anterior part of trachea, congestion of lungs and airsacculitis.

The results of bacteriological examinations are illustrated in table (1).

According to the morphology and culture characters and biochemical reactions, we could identify:

- Staph. aureus                                     3/95 isolates (2.8%).

- Staph. hyicus                                     11/95 isolates (10.4%).

- Staph. epidermis                               16/95 isolates (15.2%).

-Flavobacterium sp.                           85/95 isolates (80.7%).

-Vibrio alginolyticus.        2/95 isolates (1.9%).

On caseinate agar, highly proteolytic bacteria were classified as colonies surrounded by an inner clear zone and an outer zone of caseinate precipitates while proteolytic bacteria were identified as colonies surrounded only by caseinate precipitates. Colonies without surrounding caseinate precipitates were classified as nonproteolytic. Based on this classification, Flavobacterium and Vibrio alginolyticus are highly proteolytic gram- negative rods and Staph. aureus, Staph. hyicus and Staph. epidermis are highly proteolytic gram positive cocci.

Pathogeicity test:

Intranasal infection of 7-day–old chicks with the isolated Staph. hyicus resulted in 20-40% mortality within 6 days (p1). Before death, the birds were listless, depressed with eye affection (Fig. 1). Some birds had secretion from the eyes, mucous secretion from the nostrils and mouth breathing. PM examination showed bloody mucus in the anterior part of trachea (Fig. 2) with airsacculitis and congestion of lungs (Fig. 3). Oral infection in the 2^nd group also gave the same results. Experimental infection of 7-day-old chicks intranasally with the isolated Flavobacterium gave neither signs nor lesions but double dose of the inoculated organism (5^th group) gives mild respiratory signs with 10% moratlity. PM examination revealed bloody mucus in the trachea of some birds (Fig. 4) .

There were no signs and no lesions in the control birds. Reisolation of Staph. hyicus and Flavobacterium from trachea of the inoculated chicks was successful.

The effects of the different antibiotics on the isolated organisms are illustrated in tables
(2 and 3).

Table (1): Illustrates the results of bacteriological examinations

Table (2): Illustrates in vitro sensitivity test for Staph. hyicus

                                    Table (3): Illustrates in vitro sensitivity test for Flavobacterium

(+++) highly sensitive                                           (++) moderate sensitive                                        (+) weak sensitive                                 (-) not sensitive

Fig. (1): Experimentally infected chicks with the isolated

Staph. hyicus showing depression and eye affection

Fig. (2) Showing bloody mucus in the trachea

of experimentally infected chick with Staph. hyicus

DISCUSSION:

The proteolytic bacteria may play a role in the enhancement of Avian Influenza virus infectivity in field outbreaks where the protease enzyme which secreted by proteolytic bacteria is capable of enhancing the infectivity of influenza (Mancini et al., 2008).

Our results indicated that highly proteolytic and proteolytic bacteria are commonly present in upper trachea of poultry in varying proportions. These bacteria produces proteases that digest detectable quantities of casein in caseinate agar plates.

In our study, bacteriological examination revealed isolation of proteolytic bacteria Staph. aureus, Staph. hyicus, Staph. epidermis, Flavobacterium sp and Vibrio alginolyticus, Byrum and Slemons (1995) isolated also Staph. sciuri and Staph. xylosus beside the previous organisms.

Experimental infection of 7-day-old chicks intranasally and orally with the isolated Staph. hyicus revealed respiratory signs while intranasal infection of chicks with the isolated Flavobacterium gave no signs or lesions, while the overdose resulted in mild respiratory signs. This result is inagrrement with that recorded by Byrum and Selemons (1995). They reported that the isolated highly proteolytic bacteria from poultry may be pathogenic and associated with disease or nonpathogenic and not associated with disease.

Our conclusion proved that, highly proteolytic bacteria present in the respiratory tract of poultry may be pathogenic causing respiratory signs or non pathogenic and so the proteolytic bacteria may play a role in the enhancement of avian influenza virus infectivity in field outbreaks due to the protease enzyme, that secreted by these bacteria. So hygienic measure must be done and proper cleaning and disinfection can reduce environmental contamination by such bacteria.

REFERENCES:

Byrum B.R. and Slemons R.D. (1995): Detection of proteolytic bacteria in the upper respiratory tract flora of poultry. Avian Diseases 39 : 622–626 .

Connie, M.R. and George, M. (1995): laboratory identification of significant isolates in: Text book of Diagnostic Microbiology. Part II p. 492–499. page 530 – 531 .

Easterday, B. C., and Hinshaw V. S. (1991): Influenza in : Diseases of poultry 9^th ed . Calnek B.W., Barnes H.J., Berd, C.W, Reid, W.M. and Yoder H.W. Jr., eds. low state university press, Ames lowa. P.P. 531 – 549.

Ellen, J.B., lance, R.P. and Sydney, M.F. (1994): Methods for identification of etiological agents of infectious diseases in: Diagnostic Microbiology Edit by: Baily and Scott's.

King, M.D, Guentzel M.N., Arulanandam, B.P., lupiani, B. and chamebers J.P. (2009): Proteolytic bacteria in the lower digestive tract of poultry may affect avian influenza virus pathogenicity. Poult. Sci, 88 : 1388–1393 .

Mancini, D., Assunc, D. and portari, O. (2008): Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestions Rev. inst. Med. Trop. S. Paulo. Vol. 50, pp. 41-46 .

Merkel. J.R. (1971): Proteolytic enzyme production by Marine bacteria. Scientific Technical information Network. At the Defense Technical information Center (DTIC).

New man , J., Halvorson D., Karunakaran, D., Poss, P. and Johnson, J. (1981): Complications associated with avian influenza infection. proc. First international Avian Influenza symposium R.A. Bankowski, ed. Carter printing Co., Richmond, Va. PP.8-12 .

STUDY ON PROTEOLYTIC BACTERIA AFFECTING

THE RESPIRATORY TRACT OF CHIKENS

Hebat Allah A.E Mohamed^*, H. Kh Hassan^* and T. Y. Abd El-Motelib^**

*Animal Health Research institute, Assiut Provincial laboratory

**Dept. of poultry Diseases, Faculty of Veterinary Medicine, Assiut University

INTRODUCTION:

Easterday and Hinshaw, (1991) reported that bacteria in the respiratory flora of poultry secretes proteolytic enzymes and so are named proteolytic bacteria. These enzymes may increase the severity of avian influenza virus in poultry due to cleavage of avian influenza virus hemagglutinin molecules by proteolytic enzymes. The potential existence of this mechanism is especially appealing as an explanation for the frequent observation that many A1 viruses associated with severe clinical illness under field conditions are non pathogenic under laboratory conditions (Newman et al., 1981), and (king et al., 2009).

Byrum and Slemons (1995) showed that proteolytic bacteria were readily demonstrated among the upper respiratory tract flora of poultry included five species of Staphylococci (Staph. aureus, Staph. epidermis, Staph. sciuri, Staph. xylosus and Staph. hyicus) as well as two gram–negative bacterial species: Flavobacterium sp. and Vibrio alginolyticus, where the later 2 organisms usually reported to be associated with soil and surface water origins. The genus Vibrio found in fresh water, brackish or estuarine water and marine or salt water. The large number of proteolytic bacteria in the marine environment, coupled with the multiplicity of enzyme production by each bacterium, (Merkel 1971). Mancini et al., (2008) reported that interaction of extracellular proteases with the hemagglutinin may activate viral infection in natural infection.

The present study was under taken to cover the following points:

-Isolation and identification of proteolytic bacteria which affect the respiratory tract.

- Experimental infection of chicks with isolated proteolytic bacteria.

- In vitro sensitivity test to show the most effective drugs against the isolated organisms.

MATERIAL AND METHODS:

Material:

Specimens:

Ninety five samples (trachea) from alive and freshly dead chickens (different ages) with respiratory signs were collected from different farms of Assiut Governorate.

Media, sugars used were:

Tryptose soy agar with 5% sheep blood, caseinate agar, MacConkey's agar sodium chloride, glucose, mannose, sucrose, maltose, mannitol, lactose, esculin, yeast extract, ferric citrate, urea agar and gelatin.

Reagents:

Kovac's, oxidase and Voges – Proskauer :

Stain : Gram's stain.

Chicks for experimental infection :

Fifty, 1-day-old chicks were kept under observation for one week before the experiment.

In vitro antibiotic sensitivity discs used were:

Neomycin (30 μg), gentamycin (10 μg), ampicillin (30 μg), amikacin (30 μg), tetracycline (30 μg), spectinomycin (25 μg), streptomycin (10 μg), amoxycillin (25 μg), penicillin (10 μg), enrofloxacin (5μg), kanamycin (30 μg), cephalexin (30 μg), oxytetracycline (30 μg) and doxycycline (30 μg).

Methods:

Isolation:

Tracheal samples were inoculated on to caseinate agar and tryptose soy agar with 5% sheep blood. The bacterial culture plates were incubated aerobically at 37^oC for 24-48h. Susbected colonies were subjected to bacteriological examination to identify the organism by showing: (shape – size - colour) of the colonies, the typical morphology of the organisms by Gram's stain and studying the biochemical reactions according to Ellen et al., (1994) and Connie and George (1995).

Pathogenicity test:

Fifty, 1-day old balady chicks were used. They kept under observation for 7 days before the experiment, then they were divided as follow:

1^st group: Ten, 1-week-old chicks were inoculated intranasally with 0.5 ml of a bacterial suspension of the isolated Staph. hyicus containing 10⁶ C.FU of 24 h. culture of blood agar.

2^nd group: Ten, 1-week-old chicks. were inoculated orally with 0.5 ml of a bacterial suspension of the isolated Staph. hyicus containing 10⁶ CFU/ml of 24h. cultures of blood agar.

3^rd group: Five, 1-week-old chicks were left as control .

4^th group: Ten, 1-week-old chicks, were inoculated intranasaly with 0.5 ml of a bacterial suspension of the isolated Flavobacterium sp. containing 10⁶ CFU/ml of 24 h. culture of blood agar.

5^th group: Ten, 1-week-old chicks, were inoculated intranasally with 1ml of a bacterial suspension of the isolated Flavobacterium sp. containing 10⁶ CFU/ml of 24 h. culture of blood agar .

6^th group: Five -1week-old chicks were left as control.

In vitro antibiotic sensitivity test:

Sensitivity test were carried out on blood agar using sensitivity discs.

RESULTS:

Postmortem (PM) examination of naturally infected chickens reveald accumulation of mucus or bloody mucus in the anterior part of trachea, congestion of lungs and airsacculitis.

The results of bacteriological examinations are illustrated in table (1).

According to the morphology and culture characters and biochemical reactions, we could identify:

- Staph. aureus                                     3/95 isolates (2.8%).

- Staph. hyicus                                     11/95 isolates (10.4%).

- Staph. epidermis                               16/95 isolates (15.2%).

-Flavobacterium sp.                           85/95 isolates (80.7%).

-Vibrio alginolyticus.        2/95 isolates (1.9%).

On caseinate agar, highly proteolytic bacteria were classified as colonies surrounded by an inner clear zone and an outer zone of caseinate precipitates while proteolytic bacteria were identified as colonies surrounded only by caseinate precipitates. Colonies without surrounding caseinate precipitates were classified as nonproteolytic. Based on this classification, Flavobacterium and Vibrio alginolyticus are highly proteolytic gram- negative rods and Staph. aureus, Staph. hyicus and Staph. epidermis are highly proteolytic gram positive cocci.

Pathogeicity test:

Intranasal infection of 7-day–old chicks with the isolated Staph. hyicus resulted in 20-40% mortality within 6 days (p1). Before death, the birds were listless, depressed with eye affection (Fig. 1). Some birds had secretion from the eyes, mucous secretion from the nostrils and mouth breathing. PM examination showed bloody mucus in the anterior part of trachea (Fig. 2) with airsacculitis and congestion of lungs (Fig. 3). Oral infection in the 2^nd group also gave the same results. Experimental infection of 7-day-old chicks intranasally with the isolated Flavobacterium gave neither signs nor lesions but double dose of the inoculated organism (5^th group) gives mild respiratory signs with 10% moratlity. PM examination revealed bloody mucus in the trachea of some birds (Fig. 4) .

There were no signs and no lesions in the control birds. Reisolation of Staph. hyicus and Flavobacterium from trachea of the inoculated chicks was successful.

The effects of the different antibiotics on the isolated organisms are illustrated in tables
(2 and 3).

Table (1): Illustrates the results of bacteriological examinations

Table (2): Illustrates in vitro sensitivity test for Staph. hyicus

                                    Table (3): Illustrates in vitro sensitivity test for Flavobacterium

(+++) highly sensitive                                           (++) moderate sensitive                                        (+) weak sensitive                                 (-) not sensitive

Fig. (1): Experimentally infected chicks with the isolated

Staph. hyicus showing depression and eye affection

Fig. (2) Showing bloody mucus in the trachea

of experimentally infected chick with Staph. hyicus

DISCUSSION:

The proteolytic bacteria may play a role in the enhancement of Avian Influenza virus infectivity in field outbreaks where the protease enzyme which secreted by proteolytic bacteria is capable of enhancing the infectivity of influenza (Mancini et al., 2008).

Our results indicated that highly proteolytic and proteolytic bacteria are commonly present in upper trachea of poultry in varying proportions. These bacteria produces proteases that digest detectable quantities of casein in caseinate agar plates.

In our study, bacteriological examination revealed isolation of proteolytic bacteria Staph. aureus, Staph. hyicus, Staph. epidermis, Flavobacterium sp and Vibrio alginolyticus, Byrum and Slemons (1995) isolated also Staph. sciuri and Staph. xylosus beside the previous organisms.

Experimental infection of 7-day-old chicks intranasally and orally with the isolated Staph. hyicus revealed respiratory signs while intranasal infection of chicks with the isolated Flavobacterium gave no signs or lesions, while the overdose resulted in mild respiratory signs. This result is inagrrement with that recorded by Byrum and Selemons (1995). They reported that the isolated highly proteolytic bacteria from poultry may be pathogenic and associated with disease or nonpathogenic and not associated with disease.

Our conclusion proved that, highly proteolytic bacteria present in the respiratory tract of poultry may be pathogenic causing respiratory signs or non pathogenic and so the proteolytic bacteria may play a role in the enhancement of avian influenza virus infectivity in field outbreaks due to the protease enzyme, that secreted by these bacteria. So hygienic measure must be done and proper cleaning and disinfection can reduce environmental contamination by such bacteria.

REFERENCES:

Byrum B.R. and Slemons R.D. (1995): Detection of proteolytic bacteria in the upper respiratory tract flora of poultry. Avian Diseases 39 : 622–626 .

Connie, M.R. and George, M. (1995): laboratory identification of significant isolates in: Text book of Diagnostic Microbiology. Part II p. 492–499. page 530 – 531 .

Easterday, B. C., and Hinshaw V. S. (1991): Influenza in : Diseases of poultry 9^th ed . Calnek B.W., Barnes H.J., Berd, C.W, Reid, W.M. and Yoder H.W. Jr., eds. low state university press, Ames lowa. P.P. 531 – 549.

Ellen, J.B., lance, R.P. and Sydney, M.F. (1994): Methods for identification of etiological agents of infectious diseases in: Diagnostic Microbiology Edit by: Baily and Scott's.

King, M.D, Guentzel M.N., Arulanandam, B.P., lupiani, B. and chamebers J.P. (2009): Proteolytic bacteria in the lower digestive tract of poultry may affect avian influenza virus pathogenicity. Poult. Sci, 88 : 1388–1393 .

Mancini, D., Assunc, D. and portari, O. (2008): Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestions Rev. inst. Med. Trop. S. Paulo. Vol. 50, pp. 41-46 .

Merkel. J.R. (1971): Proteolytic enzyme production by Marine bacteria. Scientific Technical information Network. At the Defense Technical information Center (DTIC).

New man , J., Halvorson D., Karunakaran, D., Poss, P. and Johnson, J. (1981): Complications associated with avian influenza infection. proc. First international Avian Influenza symposium R.A. Bankowski, ed. Carter printing Co., Richmond, Va. PP.8-12 .