BACTERIOLOGICAL STUDIES ON THE CAPSULE OF PASTEURELLA MULTOCIDA ISOLATED FROM POULTRY IN BENI SUEF GOVERNORATE

Animal Health Research Institute, Beni Suef.

Bacteriological studies on the capsule of Pasteurella multocida isolated from poultry in Beni Suef Governorate

(With 4 Tables and 6 Figures)

By

Samia I. Afifi

(Received at 3/9/2011)

دراسات عن الغلاف الخارجى لميکروبات الباستيريلا ملتوسيدا المعزوله من الدواجن فى محافظة بنى سويف

ساميه أبراهيم عفيفى

عند اجراء الفحص البکتريولوجى لعدد (320) عينه مأخوذه من طيور نافقه ومريضه تم عزل ميکروب الباستيريلا مولتوسيدا (180) بنسبة (56.25%) وتم تعيين الخصائص الشکليه والبيوکميائيه للميکروبات المعزوله باستخدام الميکروسکوب العادى والأختبارات البيوکيميائيه وعند فحص خمس معزولات من هذه الميکروبات على أساس درجة ما أحدثته هذه المعزولات من نسبة نفوق واعراض مرضيه عند العدوى الطبيعيه بالميکروسکوب الألکترونى تم تحديد سمک الغلاف الخارجى لهذه المعزولات وکانت کالتالى:122 nm, 118 nm, 98 nm, 83nm, 81 nm, وبمقارنة نتائج الفحص المعملى لضراوة و مقاومة هذه المعزولات للسيروم کومبليمنت وقدرتها على أفراز السموم تبين ان هناک علاقه طرديه بين سمک الغلاف الخارجى لميکروبات الباستيريلا مولتوسيدا المعزوله من الداواجن المصابه بکوليرا الطيور وضرواتها و قدرتها على مقاومة السيرم کومبليمنت وقدرتها على أفراز السموم.

Summery

Bacteriological examination of 320 dead and diseased bird revealed isolation of 180 isolates of P. multocida with an incidence of 56.25%. The morphological and biochemical characters of the isolated bacteria were determined by ordinary microscope and biochemical reactions. Five isolates of P.multocida were visualizated by electron microscope on the bases of difrrence  in mortality rate, symptoms and severity of the disease in normal infection, and the  capsular thickness of these isolates were determined. The capsule thickness of the examined isolates were 122 nm, 118 nm, 98 nm, 83nm, 81 nm, the virulence,resistance to serum complement and toxigenicity of these isolates were compared experamintaly in checkins.The laboratory examination of five isolates of P.multocida with different thickness revealed that there was a correlation between the capsule thickness and the pathogenecity and toxigenicity of the examined isolates.

Key words: P.multocida, poultry, electron microscope.

Introduction

Pasteurella multocida is a gram-negative coccobacillus that is the causative agent of a wide range of diseases in animals and birds, including fowl cholera, a disease of poultry with worldwide economic importance (Carpenter et al., 1988). The organism can occur as a commensally in the naso-pharyngeal region of apparently healthy animals and it can be a primary or secondary pathogen in the disease process of birds (Tatum et al., 2005).

Kardos and Kiss (2005) cleared that fowl cholera was responsible for significant losses in poultry husbandry and Stokholm     et al. (2010) reported that the mortality rate  in commercial free-range organic layer flocks in Denmark affected by erysipelas and fowl cholera ranged from approximately 2% to 91%, with a mean of 20.8%. The pathogenesis of fowl cholera is not well understood at the molecular level, but it is likely that susceptible birds are colonized via the trachea and/or lungs, and once bacteria penetrate to the bloodstream, they multiply rapidly in the liver and spleen (Boyce et al., 2004). Toward the end stages of the disease, high levels of bacteremia often occur (Boyce et al., 2002).

Relatively little information is available on P.multocida virulence factors involved in producing fowl cholera. Because of the complex nature of bacterial pathogenesis (Rhoades and Rimler 1993). Survival of the bacteria in the blood is critical for pathogenesis, and the P. multocida capsule has been identified as the major virulence determinant that allows the bacteria to survive complement-mediated killing and to evade phagocytosis (Boyce and Adler 2000; Chung et al., 2001).

Capsules are highly hydrated polysaccharides located external and adherent to the bacterial cell wall, the locationof extracellular polysaccharides at the outermost surface of thecell is important because they are the first portal of entry andthe last barrier to excretion of substances in and out of thecell. Various hypotheses have been postulated about thefunction of the bacterial capsule, these include protection againstdesiccation in the environment (Ophir, and Gutnick 1994) phagocytosis andthe bactericidal activity of serum complement (Benchetrit et al., 1977; Boyce and Adler 2000). Boyce and Adler (2000) used electron microscope and  P.C.R. to clear that capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence in P. multocida. Borrathybay   et al. (2003) showed that the capsule thickness of avian strains of          P. multocida was correlated with their pathogenicity for chickens. Saif  et al. (2003) stated that endotoxins are produced by all P. multocida, both virulent and nonvirulent, they may contribute to virulence; however invasion and multiplication of a strain are necessary for the production of sufficient quantities of endotoxin in vivo to contribute to pathologic processes

The aim of the present work was to study the role of capsule in the pathogenesis of fowl cholera and the toxin produced by field isolates of P. multocida as virulence markers of P. multocida.

Materials and Methods

Samples:

A total number of 320 freshly dead and diseased chickens showing diarrhea and respiratory disorders were collected from different private farms at Beni-Suef Governorate from March 2007 till October 2010. The birds were submitted to bacteriological examinations. 

Isolation and identification.

Isolation and indentification of P. multocida was carried out according to CruickShank et al. (1975); Collee et al. (1996).

The internal organs of examined birds (liver, heart, spleen, lungs and trachea as well as nasal and eye discharges) were inoculated onto broth media and incubated for 18–24 h then streaked onto solid media, dextrose starch agar, blood agar, trypticase soya agar and incubated at 37°C for 24 h. Differentiation of suspected colonies were done through colonial morphology hemolysis, staining reaction, cellular morphology, biochemical reactions specially carbohydrate fermentation, indol production, motility, catalase and oxidase tests.

Bacterial strains and isolates:-

Five strains and isolates (two strains were identified by PCR in previous study in 2008 and three field isolates) of P. multocida were used to study the role of capsule in the pathogenesis of P. multocida. The strains and isolates were chosen on the bases of variation in mortality rate, symptoms, and severity of the disease in normal infection.

Electron microscopy techniques:-

Bacteria were grown in tryptic soya broth at 37°C for 18 h before growth on blood agar (tryptic soya agar containing 5% calf blood) for 18 to 24 h and prepared for electron microscopy as described by Jacques and Graham (1989)

Assessment of virulence of P. multocida for chickens:-

Sixty apparently healthy Balady chickens 4 weeks old were used to assess the virulence of P. multocida isolates for chickens, it was proved that they were free from P. multocida through cultural and serological examination, the chickens were divided into 6 groups (ten birds each) 5 groups were used to assess the virulence of five isolates of P.multocida which isolated from naturally infected chickens and group 6, (ten bird) were kept in parallel as uninfected controls. The five groups of chicken were inoculated separately intravenous with 0.1 ml of overnight broth containing 10⁹ CFU/ml of P. multocida according to Chung et al. (2001)

Serum sensitivity assays:-

The sensitivity of P. multocida strains and isolates to the bactericidal complement activity of chicken serum was determined by the addition of 10⁵ CFU to chicken serum, (serum was used at 90%) and subsequent incubation for 3 h at 41°C with aeration according to Chung et al. (2001), viable counts were determined at 0 and 3 h. Complement activity was inactivated in control samples by heating at 60°C for 30 min.

Assessment of toxin production:- according to Rhoados and Rimler (1990).

a- Preparation of endotoxin:-

Bacteria were grown in tryptic soya broth at 37°C for 18 h before growth on blood agar (tryptic soya agar containing 5% calf blood) for 18 to 24 h then inoculated into 50-ml volumes of brain heart infusion broth in 250-ml conical shake flasks and incubated at 37°C.

Culture supernates, as a source of endotoxin, were prepared from different isolates, the broth cultures were centrifuged at 7000 g for 10 min at 4°C. The resulting supernates were passed through 0.45-pm membrane filters.

b- Experimental infection:-

Thirty (one week old) balady chickens were used to assess the toxin production by P. multocida isolates, it was proved that the chickens were free from P. multocida through cultural and serological examination, the chickens were divided into 6 groups (five birds each) 5 groups were used to assess the toxin production of five isolates of P.multocida which isolated from naturally infected chickens and group 6, (five bird) were kept in parallel as controls. The five groups of chicken were inoculated separately intraperitonealy with 0.5 ml of culture supernates of P. multocida isolates.

Results

Bacteriological examination of 320 dead and diseased bird revealed isolation of 180 isolates of P. multocida with an incidence of (56.25%). The morphological and biochemical characters of the isolated bacteria was determined by ordinary microscope and biochemical reactions.

Electron microscopy techniques:-

Visualization of five isolates by electron microscope as shown in Fig. (1-6) revealed that,the capsule thickness (2-6) of P. multocida isolates were 122 nm, 118 nm, 98 nm, 83nm, 81 nm, respectively.

Fig.1. Electron micrographs of P. multocida (8000X) notice the bipolarity

Fig.2. The capsule thickness 122 nm (15 KX)

Fig.3. The capsule thickness 118 nm (30 KX)

Fig.4. The capsule thickness 98   nm (15 KX)

Fig.5. The capsule thickness 83   nm (15 KX)

Fig.6. The capsule thickness 81   nm (15 KX)

Assessment of virulence of P. multocida for chickens:-

Table 1: Survival of chickens infected with P. multocidastrains and isolates

Serum sensitivity assays:-

Table 2: The sensitivity of P. multocida strains and isolates to the bactericidal complement activity of unheated chicken serum

  Survival rate = (CFU/ml at t = 3h)/ (CFU/ml at t = 0h). 

Table 3: The sensitivity of P. multocida strains and isolates to the bactericidal complement activity of heated chicken serum

  Survival rate = (CFU/ml at t = 3h)/ (CFU/ml at t = 0h). 

Assessment of toxin production:-

Table 4: Survival of chickens injected with culture supernates of P. multocidastrains and isolates

Discussion

P. multocida is a gram-negative coccobacillus that is the causative agent of a wide range of diseases in animals and birds, including fowl cholera, that affects 100 wild avian species (Botzler, 1991). In the present study P. multocida was isolated from (56.25%) of dead and diseased chickens showing symptoms of fowl cholera Woo and Kim (2006) isolated P. multocida from two outbreaks of fowl cholera in Korea and Stokholm et al. (2010) reported that P. multocida is one of two microorganisms which produce mortalities in commercial free-range organic layer flocks in Denmark ranged from approximately 2% to 91%, with a mean of 20.8%.

Many strains of P. multocida express a capsule on their surfaces (Rimler and Rhoades. 1987). Capsules are highly hydrated polysaccharides located external and adherent to the bacterial cell wall (Sutherland, 1977). The location of extracellular polysaccharides at the outermost surface of the cell is important because they are the first portal of entry and the last barrier to excretion of substances in and out of the cell (Cheng, and Costerton. 1975). Also Snipes and Hirsh (1986), Hansen and Hirsh (1989) and Jaques et al. (1993), showed that there isa correlation between the capsule and the virulence of P. multocida. In the present study capsule of some P. multocida isolates was visualized by electron microscope and the capsular thickness of these isolates were determined and compared with their pathogenicity for chickens. The capsule thickness of P. multocida isolates as shown in Fig. (2-6) were 122 nm, 118 nm, 98 nm, 83nm, 81 nm, respectively.

Table (1) cleared that the increase of capsular thickness of P.multocida isolates the more virulence of the isolate for chicken similar result was recorded by Borrathybay et al. (2003). Regarding to table(2&3) we shown that the all tested strains were resistant to unheated serum at different levels but the survival ratio was high in heated serum the results agreed with Diallio and Frost (2000) and Chung et al. (2001), also the tables cleared that the more capsular thickness,the more virulence and the highest survival ratio, this agreed with Muhairwa et al. (2002) who stated that the most severe lesions in experimentally infected chickens were produced by a serum-resistant strain. Table (3) clearing the toxigenic activities of the examined isolates, the five isolates were toxigenic for chickens these may attributed to that the bacteria were isolated from diseased birds this result agreed with Nielsen et al. (1986); Rhoades and Rimler (1990) while Saif et al. (2003) stated that endotoxins are produced by all P. multocida, both virulent and nonvirulent, they may contribute to virulence; however invasion and multiplication of a strain are necessary for the production of sufficient quantities of endotoxin in vivo to contribute to pathologic processes. In conclusion capsule of P. multocida is a major virulence determinant that allows the bacteria to survive complement-mediated killing and to evade phagocytosis which leading to multiplication of the bacteria and consequently produce the endotoxin, it is an essential virulence factor.

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