Dept. of Pathology and Clinical Path. 

Fac. Vet. Med., Assiut Univ., Assiut, Egypt

Studies on Pasteurella multocida and other bacterial pathogens associated with some problems in duck farms

in Assiut Governorate

(With 2 Tables and 8 Figures)

By

K. Radad and Fatma A. Moustafa*

*First researcher in Assiut Animal Research Center

(Received at 20/12/2005)

دراسات عن الباستيريلا ملتوسيدا وبعض البکتريا المرضية المصاحبة

لبعض المشاکل فى مزارع البط بمحافظة اسيوط

خالد رداد ، فاطمة عبد المجيد مصطفى

عند استقصاء مدى تواجد ميکروب الباستيريلا ملتوسيدا والميکروبات المصاحبة لها فى بعض مزارع البط الحکومية والخاصة بمحافظة اسيوط ، تم اخذ عينات من الطيور المريضة اکلينيکيا وذلک من الاعضاء المحتوية على علامات باثولوجية للعدوى البکتيرية مثل الکبد والقلب والقصبة الهوائية والرئتين والطحال. خضعت جميع العينات للفحوص البکتيرية والفيروسية والميکوبلازما. تم عزل عدة عوامل بکتيرية وکانت النسبة الکلية للبکتريا المعزولة (%51.7) ، باستيريلا ملتوسيدا (%25) ، الميکروب القولونى (%16.7) ، کامبيلوباکتر جوجيناى (%3.3) ، باستيريلا ملتوسيدا + الميکروب القولونى (%6.7 ). ومن ناحية اخرى أعطت المحاولات المبذولة للعزل الفيروسى والميکوبلازما نتائج سلبية. تم إجراء العدوى الصناعية فى بط عمر 3 أسبوع بالبستيريلا ملتوسيدا. هذا وقد تم ملاحظة الأعراض الاکلينيکية والصفة التشريحية التى تشبه إلى حد کبير تلک التى لوحظت فى العدوى الطبيعية بعد 72 ساعة من العدوى. تم ايضا عزل البستيريلا ملتوسيدا مرة أخرى من الطيور المصابة تجريبيا. عند اجراء اختبار الحساسية للمعزولات معملياً ضد بعض المضادات الحيوية، قد تبين ان جميع المعزولات شديدة الحساسية لکل من الجنتاميسين والانروفلوکساسين والاوکسيتتراسيکلين. بالاصافة الي ذلک تم اخذ بعض العينات من الطيور المصابة تجريبيا للفحص الهستوباثولوجى الذي اسفر عن وجود بعض الافات الباثولوجية في کل من الکبد والقلب والمخ والرئتين. ومن تلک الدراسة يتضح مدى تواجد ميکروب البستيريلا ملتوسيدا کمسبب لبعض المشاکل المرضية فى مزارع البطبمحافظة اسيوط ومدى حساسيته لبعض المضادات الحيوية.

Summary

Pasteurella multocida (P. multocida) and several bacterial agents associated with some problems in ducks were investigated in different governmental and private farms at Assiut Governorate. Samples were taken from different tissues showing pathological evidence of infection such as liver, heart, trachea, lungs and spleen as well as from blood. All samples were subjected for bacterial, mycoplasma and viral isolation. It was found that the total percentages of isolated bacteria were (51.7%), P. multocida (25%), E. coli (16.87%), Campylobacter jejuni (3.3%), and mixed infection with P. multocida and E. coli (6.7%). On the other hand, all examined samples were negative for mycoplasma and viruses. Moreover, experimental infection was carried out with isolated P.multocida in two weeks old ducks. After 72 h of infection, samples were taken for both bacterial isolation and histopathological examination. P. multocida was also isolated from experimentally-infected ducks. When they subjected to the in vitro sensitivity tests, it was shown that they were highly sensitive to gentamycin, norfloxacin and oxytetracyclin. Among examined tissues of experimentally-infected ducks, liver, heart, brains and lungs showed multiple pathological changes. Thus, the present study has shown the role of P. multocida as a causative agent of some problems in duck farms at Assiut Governorate and their sensitivity for some antibiotics.

Key words: Pasteurella multocida, duck, pathology, pathogenicity

Introduction

Avian cholera is a highly contagious disease caused by the gram-negative bacterium P. multocida which is responsible for widespread disease that affects about 100 wild avian species (Botzler, 1991). Epizootics caused by P. multocida occur almost in waterfowl populations and cause annual mortality in various waterfowl areas (Samuel et al., 2003). Avian cholera usually appears as a septicemic disease results in high morbidity and mortality rates among affected birds. Sometimes, the disease occurs in the form of epornitics due to rapid spread and extraordinary virulence of the organism (Samuel et al., 2003). The important sources of infection with P. multocida in waterfowl are including carrier birds and wetland sediments and water. Also, contamination of the environment particularly water after epizootics facilitates transmission of P. multocida among dense populations of waterfowl either by ingestion and/or the inhalation of aerosols (Botzler, 1991). Additionally, bird-to-bird contact may be considered an important component of transmission (Wobeser, 1992).

Regarding P. multocida infection in ducks, it was reported that pasteurellosis is an important septicemic disease of growing ducklings (Gordan and Jordan, 1982). For instances, Bayoumi et al. (1988) reported epornitics of pasteurellosis in duck farms caused 5-30% mortalities with higher rates in birds of more than 4-weeks of age. Rhoades and Rimler (1988) reported that P. multocida infection is a disease of young ducklings and ducks younger than two weeks old usually die 1-2 days after appearance of clinical signs and older birds may survive a week or more. Moreover, Ibrahim (1991) isolated P. multocida from ducks over 10 weeks of age. Nakamine et al. (1992) and Takahashi et al. (1996) recovered P. multocida from 25% of 200 Muscovy ducks in a farm died of an acute disease in Japan. Mariana and Hirst (2000) isolated 10 strains of P. multocida and two strains of P. gallicida and P. septica from ducks in Indonesia.

Clinical signs of P. multocida infection in ducks were including ocular and nasal discharges, mild coughing, greenish diarrhea, ataxia, tremors of head and neck followed by coma (Chaudhury and Mahanta, 1985). Subacute disease was characterized by lameness, corneal turbidity, dystasia and depression (Takahashi et al., 1996).

Necropsy findings due to acute P. multocida infection in ducks were congested lungs and enlarged liver and spleen (Gordon and Jordan, 1982). In less acute disease, fibrinous pericarditis, periheptatitis, airsaculitis and meningitis were observed (Rhoades and Rimler, 1988). Histopathologically, ducks died acutely of avian cholera showed lesions of hemorrhagic septicemia with widespread vascular damages and focal necrosis in the liver and other organs (Hunter and Wobeser, 1980; Nakamine et al., 1992). Moreover, Songserm et al. (2003) reported that P. multocida played a role in induction of sinusitis in ducks as they isolated P. multocida, group B and serotype 3, from sinusitis in the affected ducks.

This work deals with the investigation of the cause of death of some outbreaks suspected to be due to P. multocida and some associated infectious agents in duckling in some governmental and private farms at Assiut Governorate. Investigations were done through isolation and identification of the etiological agents using morphological and biochemical properties and studying the pathogencity of P. multocida to experimentally-infected ducklings. Moreover, the in vitro sensitivities of the isolated P. multocida strains to different chemotherapeutic agents were also studied.

Materials and Methods

A total of 120 freshly dead and sacrificed ducklings (3-10 weeks old) were obtained from different governmental and private farms at Assiut Governorate. Those birds showed depression, diarrhea and morbidity and mortality rates reached to 10 and 25%, respectively. Clinical signs manifested by diseased birds and necropsy findings encountered in dead birds were studied and described.

Experimental infection

Experimental infection was done for testing the pathogenicity of the recovered P. multocida from naturally infected birds. Twenty five ducklings (3 weeks old) were obtained from the Farm of Faculty of Agriculture, Assiut University, Assiut for testing. It was proved that they were free from P. multocida infection through cultural and serological examination.

Isolation of etiologic agents

a) Bacterial isolation

Bacterial isolation was carried out from different organs of freshly dead ducklings including liver, heart, lungs, trachea and spleen as well as from nasal and eye exudates. Isolates were inoculated into tryptose broth, brain heart infusion broth, nutrient broth and Campylobacter enrichment broth. Tryptose broth and nutrient broth tubes were incubated at 37°C for 18-24 h. Brain heart infusion broth was incubated at 31°C for 3 days. Campylobacter enrichment broth was incubated at 42°C for 48 h in an atmosphere of 5% O₂, 10% CO₂ and 5% N₂ by using an anaerobic jar and Campylobacter gas generating kits. Thereafter, subculturing was done on solid media.

P. multocida examination

For characterization of P. multocida, blood agar or dextrose starch agar plates as selective medium for P. multocida were used and incubated at 37°C for 24 h. They were examined for non-hemolytic and dew drop like colonies. Suspected colonies were cultured in broth for 6 h at 37°C for storage at -80°C (Lee et al., 1991; Rimler and Glisson, 1997).

E. coli examination

Broth cultures were spread on MacConkey’s agar and incubated at 37°C for 24 h. Suspected E. coli colonies were selected and purified. Then, pure colonies were picked and stained by Gram stain (Cruick-Shank et al, 1975).

Campylobacter jejuni examination

Campylobacter blood agar base supplemented with skirrow Campylobacter selective supplement were incubated at 42°C for 48 h in microaerobic atmosphere of 5% O₂, 10% CO₂ and 5% N₂ using Gas-pak anaerobic jar and Campylobacter gas generating kits (Skirrow and Benjamin, 1980; Aarestrup et al., 1997).

Mycoplasma examination

Brain heart infusion agar plates were incubated at 37°C in moist candle jar under reduced oxygen tension for detection of mycoplasma according to Sabry and Ahmed (1975) and Ball et al. (1994).

Identification of isolates

Identification of different microorganisms was carried out according to Cruick-shank et al. (1975) and Collins and Lyne (1991) as followings:

1- Cellular morphology.

2- Colonial morphology including color, shape, size, odor and pigment production.

3- Biochemical reactions such as carbohydrate fermentation test using sugar media (1% peptone water + dextrose, sucrose, lactose, maltose), indole production, catalase test, urease test, citrate utilization, motility, MacConkey’s agar growth and hemolysis on blood agar.

b) Virus isolation

Bacteria free suspension was prepared from tissues obtained from liver, spleen and kidneys in antibiotics-containing phosphate buffered saline. Samples were centrifuged at 4000 rpm for 20 min. The supernatant was inoculated into allantoic sac of 10 day old embyronated chicken eggs and incubated for 6 days. Embryos were candled daily and fluids were harvested and tested for haemagglutination activity (Yamaguchi et al., 1981).

Pathogenicity test in mice

Five mice of 3-4 weeks old were obtained from Animal House Unit, Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut. Animals were inoculated subcutaneously with 0.2 ml of 10⁶ cfu of P. multocida obtained from diseased birds. Mice died      24-48h after inoculations were subjected to postmortem examination. Re-isolation of P. multocida was carried out from infected mice (Cruick-shank et al., 1975).

Pathogenicity test in ducklings

Twenty five healthy ducklings of 3 weeks old were used for testing the pathogenicity of recovered P. multocida from naturally infected birds. Birds were inoculated intramuscularly with 0.1 ml of overnight broth 7x10⁷ P. multocida/duck. Five ducklings were kept in parallel as uninfected controls (Ibrahim, 1991; Pehlivanoglu et al., 1999).

Antibiotic sensitivity test

The sensitivity of isolated P. multocida to different antibiotics was done by Disc diffusion method using dextrose starch agar according to Cruick-shank et al. (1975). Methods according to Finegold and Martin (1982) using Muller Hinton agar and Bopp et al. (1985) using Brucella agar base supplemented with 5% sheep blood were used for E. coli and Campylobacter jejuni, respectively. Used antibiotic discs were ampicillin (10 µg), streptomycin (10 µg), gentamycin (10 µg), chloramphenical (30 µg), nalidixic acid (30 µg), norfloxacin (5 µg), erythromycin (15 µg), neomycin (30 µg), colistin sulphate (25 µg) and oxytetracycline (30 µg).

Histopathology

Tissue specimens were taken from liver, heart, brain and lungs of experimentally-infected ducklings. Specimens were fixed in 10% neutral buffered formalin and processed routinely for paraffin embedding techniques. Embedded tissues were sectioned at 3 um and stained with hematoxylin/eosin (Bancroft and Stevens, 1982).

Results

Clinical examination

Diseased ducks of different ages showed depression, ruffled feathers, anorexia, mucous discharges from body orifices and diarrhea. Necropsy findings were represented by congestion of the carcasses, necrotic foci and multiple petichiae on liver and spleen, congestion and edema of the lungs and hemorrhages on the intestine particularly the duodenum.

Bacterial isolation

Among 120 samples from different organs of duck flocks, 62 samples (51.7%) were found positive for bacterial isolation. It was also found that 30 sample (25%), 20 samples (16.7%), 4 samples (3.3%) and 8 samples (6.7%) were positive for P. multocida, E. coli, Campylobacter jejuni and mixed infection of P. multocida and E. coli infections, respectively (Table 1).

Identification of isolated organisms

P. multocida

When P. multocida examined morphologically, they appeared as gram negative, short cocco-bacilli, non-motile and bipolar-stained organism. Biochemically, the organism was non-hemolytic on blood agar and did not grow on MacConkey’s agar. Moreover, all isolates fermented dextrose, sucrose, mannitol and galactose. But they did not ferment maltose, lactose and arabinose. When the isolates were injected subcutaneously into both mice and ducklings, they produced characteristic lesions for P. multocida.

E. coli

Isolated strains of E. coli were observed as gram negative bacilli, smooth glossy and rose-pink in color on MacConkey’s media.

Campylobacter jejuni

Campylobacter strains appeared as small, gram negative and curved or spiral rods. They were motile in semi-solid Brucella agar, non-hemolytic when grown on Brucella blood agar and grown at 42°C. They were also oxidase and catalase positive, hydrolyse hippurate and produced inhibition zone of 12-14 mm in nalidixic acid sensitivity test.

Mycoplasma detection

There was no evidence for mycoplasma infection in examined samples.

Viral isolation

No viral agents were recovered from the examined specimens.

Pathogenicity of P. multocida in ducklings

Clinical symptoms in experimentally-infected ducklings with P. multocida appeared 72 h after intramuscular inoculation. They were including emaciation, drowsiness, fever, respiratory signs, lacrimation and dysponea. Birds exhibited morbidity rate about 80% and mortality rate reached 75%. Postmortem lesions were arthritis, congestion of the liver, spleen and lungs and peticheal hemoorhage on the liver and coronary fat with presence of minute necrotic foci on the liver surface. Surviving birds showed incoordination and ataxia. Re-isolation trials were adopted from liver and heart. No lesions were observed in control birds which found also negative for isolation of the organisms.

Antibiotics sensitivity test

The in vitro sensitivity test showed that the three isolated microorganisms were highly sensitive to gentamycin, norfloxacin and oxytetracyclin. The obtained results were illustrated in Table (2).

Histopathology

The most common histopathological findings were observed in the liver, heart, brain and lungs. HE-stained hepatic sections revealed congestion and dilatation of blood sinusoids with red blood cells (Fig.1), vacuolar degeneration of the hepatocytes (Fig. 2), focal areas of necrosis (Fig. 3) and mononuclear cellular infiltration in the portal areas (Fig. 4). The lesions in the heart were including congestion of blood vessels, interstitial edema and degeneration of the myocardial fibers (Fig. 5). There were congestion of blood vessels, perivascular edema and degeneration of some neurons in the brain (Fig. 6). Lungs showed congestion and thrombosis of blood vessels (Fig. 7), interstitial edema and necrosis of the epithelial lining of parabronchiols (Fig. 8). 

Table 1: Incidence of bacterial isolates from examined ducks.

Table 2: Antibiotic sensitivity test for P. multocida, E. coli and Campylobacter jejuni isolates from ducks.

+++         High sensitivity.                                      +      Low sensitivity.

++           Intermediate sensitivity                         R      Resistant.

Discussion

Duck cholera is considered the most important bacterial disease threatens duck breeding in Egypt (Gorgy, 1992) and there were some recent reports discussed the role of P. multocida as a causative agent of the disease in ducks (Samuel et al., 2003; Songserum et al., 2003).

In the present study, clinical signs and gross necropsy findings manifested by diseased and dead birds were similar to those described by Rhoades and Rimler (1988) and Ibrahim (1991), respectively. They were including marked depression, anorexia, diarrhea and mucous discharges from the body orifices. The postmortem findings were congestion of the carcasses, petechial hemorrhages through out the viscera and necrotic foci on liver surface, edema and pneumonia in the lungs. 

When different samples from liver, heart, trachea, lungs, and spleen subjected for bacterial isolation, gram negative, bipolar, non-motile, non spore forming and non-hemolytic bacteria which also did not grow on MacConkey’s agar was isolated. In parallel, Lee et al. (1991) and Rimler and Glisson (1997) reported agreement results. Moreover, all isolates had sugar fermentation pattern and biochemical properties similar to the general characteristics of P. multocida reported by Rhoades and Rimler (1991) and Ibrahim (1996).

P. multocida was isolated from ducks at rate of 25%. Similarly, El-Ghawas (1975), Nakamine et al. (1992) and Takahashi et al. (1996) isolated P. multocida form outbreaks in Muscovy ducksat the same rate. Lotfy et al. (1970) isolated P. multocida from ducks at a higher rate reaching 42.5%. E.coli and Campylobacter jejuni were isolated in association with P. multocida. They were isolated at rate of 20% and 3.3%, respectively. These results were in consistent with the finding of Harbourne (1962), Songserm et al. (2003) and Dobbin et al. (2005) who reported that P. multocida infection was nearly always accompanied by E. coli and Campylobacter spp. infection in free-living fowls. Fallacara et al. (2001) isolated E. coli at the rate of 67% which much higher than our results.

To reveal the pathogenicity of P. multocida, isolates from naturally-infected cases were inoculated intramuscularly in 3 weeks old ducklings. The clinical signs, mortality rate and necropsy findings appeared 72 h post-inoculation and were characteristic for P. multocida infection according to Ibrahim (1991) who recorded mortality rate of 80%.

Histologically, dead birds showed marked vascular findings in the liver, heart, brain and lungs. They were including congestion of blood vessels, hemorrhages, thrombosis and edema. In consistent, Hunter and Wobeser (1980) reported prominent vascular lesions in experimentally-infected mallard ducks with P. multocida. Hunter and Wobeser (1980) attributed these vascular changes to endotoxin-initiated disseminated intravascular coagulation. Other findings were vacuolar degeneration and necrosis of hepatocytes and mononuclear cellular infiltration in the portal areas in the liver, degeneration of the myocardial fibers in the heart, degeneration of some neurons in the brain and necrosis and sloughing of the epithelial lining of parabronchiols. These lesions were milder than those reported by Hunter and Wobeser (1980) and this may be due to species and age differences. Also, the route of inoculation was also participating (Pehlivanoglu et al., 1999). 

P. multocida strains were highly sensitive to gentamycin and oxytetracyclin and highly resistant to ampicillin, erythromycin, chloramphenicol and neomycin. These finding are in agreement with the observation of Shaw et al. (1990), Nakamine et al. (1992) and Takahashi et al. (1996). As illustrated in Table (2), E. coli isolates were susceptible to gentamycin, norflaoxacin, colistin sulphate and oxytetracyclin and were resistant to ampicillin, streptomycin, chloramphenical, nalidicic acid and Erythromycin. Erganis et al. (1989), Moharana et al. (1993) and Jakeen et al. (1999) reported agreement results. Campylobacter jejuni strains were highly sensitive to gentamycin, norfloxacin, ertythromycin and oxytetracyclin and resistant to ampicillin, streptomycin and colistin sulphate. These finding are in agreement with the results of Nakai et al. (1994) and Das et al. (1995).

In conclusion, bacterial isolation from freshly dead and sacrificed ducks in some governmental and private farms at Assiut Governorate revealed that the total percentages of isolated bacteria were (51.7%), P. multocida (25%), E. coli (16.87%), Campylobacter jejuni (3.3%) and the mixed infection with P. multocida and E. coli (6.7%). On the other hand, all examined samples were negative for mycoplasma and viruses. Experimental infection with isolated P. multocida in three weeks old ducklings produced the same characteristics of P. multocida infection 72 h post-infection. The in vitro sensitivity tests have shown that isolated P. multocida was highly sensitive to gentamycin, norfloxacin and oxytetracyclin. The present study has shown the role of P. multocida as a causative agent of some problems in duck farms in Assiut Governorate and their sensitivity for some antibiotics.

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