BACTERIOLOGICAL AND PATHOLOGICAL FINDINGS ON PASTEURELLOSIS IN OSTRICHES

Dept. of Avian Diseases,

Animla Health Research Institute, Dokki

Bacteriological And Pathological Findings On Pasteurellosis In Ostriches

(With 5 Tables and 16 Figures)

By

A.M. Shalaby and Amal I. Yoseif

 (Received at 12/6/2008)

دراسة بکتريولوجية وباثولوجية على عدوى الباستيريللا فى النعام

محمود حمدى شلبى ، أمال ابراهيم يوسف

فى محاولة لأکتشاف دور الباسيتريلا مالتوسيدا فى المشکلات المرضية فى مزارع النعام فى مختلف المحافظات فى مصر تم فحص 22 حالة مرضية ممثلة فى 154 عينة من هذه الحالات (کبد - قلب – رئة - طحال - نخاع العظمى - قصبة هوائية) بالاضافة الى مسحات أنفية من الطيور المريضة وقد أوضحت الأعراض ارتفاع نسبة النفوق والأصابة الأضافة الى أعراض تنفسية وهضمية. وقد أثبتت الصفة التشريحية صورة التسمم الدموى بالاضافة الى الالتهاب المعوى. وأسفر الفحص البکتيرى عن عزل 42 معزولة من 154 عينة بنسبة عزل مقدارها 27,3%. وصنفت العترات سيرولوجيا الى 5:A, 8:A , 9:A,2:D بينما 12 معزولة لم تصنف , ووجدت جميع المعزولات شديدة الضراوة للفئران والدجاج. أثبتت اختبارات الحساسية للمعزولات شدة حساسيتها للسيبروفلوکساسين التراى ميثوبريم والکولستين سلفات والدوکسيسيکللين ومقاومتها للبنسللين واللينکومايسين. بينما أثبتت    التغيرات الباثولوجية للأعضاء الداخلية المختلفة شدة ضراوة التغيرات الباثولوجية. وفى المجمل فقد ثبت أن الباسيتريلا مالتوسيدا شديدة الضراوة لمزارع النعام مسببة خسائر فادحة. کذلک وجد ارتفاع نسبة عزلها بالنسبة للأنواع الأخرى من البکتيريا لذلک يلزم اتباع قواعد  الامان الحيوى فى مزارع النعام لتقليل نسبة العوامل المساعدة على الاصابة مع اتباع القواعد الصحية الصارمة فى مزارع وأماکن تربية ومجازر النعام للتحکم فى الاصابة بهذا الميکروب.

SUMMARY

In a trial to investigate the role of Pasteurella multocida in disease problems in ostrich farms at different provinces in Egypt. Twenty two cases are obtained and 154 samples from these cases (liver, heart, spleen, lung, trachea and Bone marrow) beside Nasal cleft swab from diseased birds. The clinical signs revealed high morbidity and mortality with respiratory distress and gastroenteric findings. The post mortem findings revealed a picture of hemorrhagic septicemia with enteritis. Bacteriological Examination revealed isolation of 42 isolates from 154 samples with a percentage of 27.3%. The serotyping of the isolated strains revealed that they belong to serotypes 5: A, 8: A, 9: A and 2: D while 12 isolated strains were untyped. All the isolated strains were highly pathogenic for mice and chickens. The antibiogram test revealed that highly sensitive to ciprofloxacin, trimethoprim, colistin sulphate and doxycycline and resistant for penicillin, lincomycin. The pathological changes in different internal organs revealed highly pathological changes. It could be concluded that Pasteurella multocida is highly pathogenic for ostriches and cause severe losses in ostrich farms and found that the rate of isolation of Pasterulla multocida is very high in most bacterial agents. Therefore biosecurity measures must be completely used to minimize the predisposing factors with strict hygienic measures at the farm slaughter house and back yard of ostrich farms.

Key words: Ostrich, pasteurellosis, P. multocida

INTRODUCTION

Pasteurellosis in commercial poultry is usually associated with Pasteurella maltocida. There is many documented cases of Pasteurellosis in ostriches (Hala and Kawther, 2006; Moursi and Husein, 2007; Fatma and Hala, 2008).

  Pasteurelosis occurs extensively in backyard and ornamental poultry. Ostriches have become commercially farmed animals in several countries of the world. Since 1999, there has been a dramatic increase in the number of ostriches. Although some technical information is available on the necessary conditions required for the successful rearing of ostrich chicks under farming conditions (Deeming and Ayres,1994) problems still exist that are characterized by variations in growth rate which can adversely affect the value of the bird for breeding or slaughter. Infectious and nutritional diseases are some of the constraints to available ostrich. Ostriches are susceptible to numerous diseases of bacterial, fungal or parasitic origin. For some diseases affecting ostriches, new methods of control are poorly documented and an attempt was made to apply methods used in poultry biosecurity to ostriches (Huchzer meryer, 1996 and Cooper 2005). There are no effective vaccination protocols for ostriches and ideal standards of hygiene and quarantine are needed to prevent introduction of infection into ostrich flocks (Tully and Shane, 1996).

The aims of the present study were:

1 – Isolation and identification of P.multocida from ostriches with studying its pathogenicity and antibiotic sensitivity to various antibiotics.

2- Studying the histopathological changes associated with Pasteurellosis in ostriches.

MATERIALS and METHODS

Collected samples:

A total of 10 ostriches farms were examined from different governorates in Egypt (Alexandria, Sharkia, Ismaelia and kalubia) for the presence of  Pasteurella infection (Table 1). Samples were collected from organs of ostrich both freshly dead and slaughtered.

Histopathological examination:

Specimens of trachea, lung, liver, proventriculus, pancreas, kidney and spleen were taken from the diseased ostriches and fixed in 10% neutral buffered formalin. The fixed specimens were trimmed, washed dehydrated in different concentrations of alcohols, cleared in xylol and embedded in paraffin. The paraffin embedding blocks were cut into sections sum and stained with Haematoxylin and Eosin (H&E) according to Bancraft et al. (1990).

Bacteriological examination:

The collected samples included, heart, liver, spleen, bone marrow and nasal cleft swabs were submitted to Poultry Diseases Department, Animal Health Research Institute, Dokki, Giza, in which the case history, clinical signs and postmortem examination were recorded.

All the collected samples were examined bacteriologically according to(Snipes et al., 1990) as follow: the collected samples were inoculated in brain heart infusion broth (Oxoid) incubated at 37°C for 24 hrs. Direct cultivation of different samples on blood agar, Nutrient agar and MacConkey agar plates and incubated at 37°C for 24hrs. A loopful of each broth culture were inoculated on blood agar, MacConkey agar plates (Oxoid) and incubated at 37°C for 24hrs. Isolated colonies were picked up and identified morphologically, microscopically and biochemically according to (Holt et al., 1996).

Pathgenicity testing of each of the suspected isolates was carried out by inoculation of two mice I/P with 0.2 ml of over night broth culture of the suspected P. multocida isolates. Blood smears from dead mice were stained with Giemsa stain for detection of Pasteurella bipolarity and inoculated on blood agar plates for re-isolation of the microorganism.

Serological identification:

Capsular typing of involved strains were determined by indirect haemagglutination test according to Carter and Rappay (1962) using diagnostic type specific Pasteurella maltocida antisera (type A, B, D and E).

Antibiotic susceptibility tests:

In-vitro susceptibility testing of P. multocida isolates to various antimicrobial agents was determined against twelve different commercial antibiotic discs (Oxoid) according to Quinn et al. (1994). 

Pathogenicity tests of P. maltocida isolates:

1- In mice:

A total of fifty Swiss mice were used for pathogenicity testing of the isolated serovars which were proved to be free from pasteurella infection. The mice were divided into five equal groups (10 mice per group). Mice in groups number 1, 2, 3 and 4 were infected intraperitoneally (I/P) with 0.2 ml of overnight broth culture of one of the isolated pasteurella serovars (5: A, 8: A, 9: A and 2: D respectively) while mice in group (5) were kept as control non-infected group. Mice in each group were monitored for clinical signs and mortality for 7 days and deaths were recorded.

2- In chickens:

A total of fifty, 8 weeks old white leghorn chickens and suitable balanced ration were brought from commercial farm. Chickens were proved to be free from Pasteurella infection. Chickens were divided into 5 equal groups (10 chickens per group). Chickens in groups number 1, 2, 3 and 4 were infected  intra- nasal  with 0.2 ml of overnight broth culture of one of the isolated Pasteurella serovars (5:A, 8:A, 9:A and 2:D respectively) while chickens  in group (5) were kept as control non-infected group. Chickens in each group were monitored for clinical signs and mortality for 7 days and deaths were recorded.

RESULTS

1- Clinical signs:

Among the affected birds were varied according to the severity of the disease, but generally these were gastrointestinal troubles manifested by diarrhea with offensive odor- other signs as emaciation, dullness, inappetence and recumbency before death were recorded. Respiratory distress and dyspnea were also common with nasal discharge as well as conjunctivitis.

2- Postmortem findings:

It revealed a picture of septicemia congestion of the liver, lung and spleen with enlargement of the heart and thick pericardium were seen severe congestion of the intestine. Bacterial isolation and Identification revealed that Pasteurella multocida isolated from different provinces (Alexandria, Sharkia, Ismaelia and Kaluobia) with the percentage of (28.6%, 31.4%, 28.6% and 52.4%) respectively according to the number of samples from each of them. Table (1) the percentage of the isolated Pasteurella multocida from different samples of ostrich birds were reported in Table (1).

-          The serotyping of the isolates revealed that 5: A, 8: A, 9: A, 2 : D and untype isolates were reported in Table (2).

-          The pathogenicity tests of the isolated strains were more pathogenic for mice and chicken. The results are shown in Table (4) and (5).

-          The results of antibiogram sensitivity test are mentioned in Table (3).

-          In the present study a wide spread pathological changes in different organs are recorded.

In the present study:

Therefore a wide spread pathological changes in different organs were recorded. It could be concluded that the most bacterial pathogens which detected in ostrich farms were Pasteurella multocida so; successful management and disinfection of farms must be carried with therapy to eliminate the predisposing factor as much as possible.

Pathological lesions of internal organs of freshly dead ostriches:

 Trachea, the surface epithelium of lamina propria showed hyperplasia (Fig. 1). Thickening of the mucosa due to edema,congested blood vessels, haemorrhage and inflammatory cells  infelteration (Fig. 2).

Lung lesions consisted of haemorrhage. (Fig. 3), diffuse infelteration and focal aggregation of mononuclear cells mainly lymphocyte, fibrinous component is most prominent (Fig. 4).

Liver, the hepatic vessels of most cases showed congestion with subcapsuler haemorrhage. Some of the hepatic cells suffered from vaculer and granular degeneration in addition to activation of kuffer cells Fig. 5). Haemorrhage was seen (Fig. 6). Somstimes the portal area exhibited inflammatory cells aggregation, some cases revealed coagulativ necrosis.

Kidney lesions consisted of moderate congestion of blood vessels and haemorrhage (Fig. 7), The renal tubules suffered from necrobiotic changes, epithelial denudation and granular degeneration (Fig. 8), lymphocytic aggregation between the tubules was exist. Necrosis of renal tubules which replaced by inflammatory cells (Fig. 9) and edema in ureter (Fig. 10).

Spleen, microscopical changes revealed severe haemorrhage, haemosedrosis (Fig. 11) and multipl focal necrosis of apleomorphic cells (Fig. 12).

Heart lesions consisted of pericarditis, congestion of blood vessels haemorrahages and dispertion of myelocytes due to edema and inflammatory cells infiltration (Fig. 13) and myomalacia.

Proventriculus, showed hyperplasia of epithelial lining, congestion and inflammatory cells infiltration in mucosal layer. There was degeneration of glandular epithelium, haemorrhage and lymphocytic infiltration (Fig. 14).

Pancrease, showed necrobiotic changes (Fig. 15) and haemorrhage (Fig. 16). 

Table 2: Incidence of isolation of P. multocida serovars from ostriches cases in different Governorates.

Table 3: Antibiotic susceptibility tests of the 42 P. maltocida isolates from ostriches:

Table 4: Pathogenicity of P. multocida isolated from ostriches in mice

Table 5: Pathogenicity of P. multocida isolated from ostriches in chickens:

Histopathological figures

Fig. 1: Trachea of diseased ostrichs showing hyperplasia of epithelial lineing lamina propria.                                           (H-E x100)

Fig. 2: Trachea of diseased ostrichs showing edema and haemorrhage in mucosa.                                                               (H-E x100)

Fig. 3: Lung of diseased ostrichs showing haemorrhage.        (H-E x250)

Fig. 4: Lung of diseased ostriches showing fibrinous exudates.

                                                                                               (H-E x100)

Fig. 5: Liver of diseased ostriches showing vacular degeneration.  

                                                                                               (H-E x400)                                                                                                        

Fig. 6: Liver of diseased ostriches showing hemorrhage and coagulative necrosis.                                                               (H-E x100)

Fig. 7: Kidney of diseased ostriches showing hemorrhage.     (H-E x250)

Fig. 8: Kidney of diseased ostriches showing denudation of renal  tubule.                                                                  (H-E x250)                                                                     

Fig. 9: Kidney of diseased ostriches showing necrosed tubules which replaced by inflammatory cell.                                 (H-E x100)

Fig. 10: kidney of diseased ostriches showing edema in ureter.

                                                                                               (H-Ex100)

Fig. 11: Spleen of diseased ostriches showing severe haemorrhage.

                                                                                              (H-E x100)

Fig. 12: Spleen of diseased ostriches showing multiple focal necrosis of             pleomorphic cells.                                                  (H-E x400)

Fig. 13: Heart of diseased ostriches showing dispertion of myelocytes due to edema and inflammatory cells.                    (H-E x 250)

Fig. 14: Proventriculus of diseased ostriches showing degeneration of glandular epithelium and hemorrhage with lymphocytic infiltration.                                                             (H-E x100)

  Fig. 15: Pancreas of diseased ostriches showing necrobiotic changes.

                                                                                              (H-E x250)

Fig. 16: Pancreas of diseased ostriches showing hemorrhage.  (H-Ex250)        

DISCUSSION

The growing interest in commercial production of ostriches all over the world including Egypt has accompanied progress in researches concerning diagnosis and control of ostrich diseases. Young chicks are more susceptible to bacterial infection and parasitic diseases associated with relatively high mortality (Huchzermeyer, 1999, Tully, 2000 and Cooper, 2005).

The clinical signs of affected ostriches were varied according to severity of the disease in affected birds but generally respiratory distresses and dyspnea were recorded with ocular and nasal discharge, conjunctivitis. The results agreed with Sohair and Moursi (2003) and Sahinduran (2004). Gastrointestinal troubles which were manifested by diarrhea with offensive odor. Poor growth, emaciation and recumbencey before death were observed. These clinical observation were similar to those reported by Tully and Shan (1998); El Demerdech et al. (2003); and Moursi and Husien (2007).

Post-mortem examination revealed a picture of septicemia, congestion of the liver, lung, and spleen with enlargement of the heart. Similar post mortem picture was recordedby Okoh (1980), Tully and Shan (1998), El- Demerdach et al (2003) and Moursi and Husien (2007).

Bacteriological examination revealed that Pasteurella multocida were isolated with percentage of 27.3% in our study but Nasef et al. (2003) who reported the Pasteurella multocida with a percentage of 6.61%.

The incidence of bacterial isolate from diseased and dead birds was very high in Pasteurella multocida. This result confirms that obtained by El-Faki et al. (2002) and Sohair and Moursi (2003) who reported the isolation Pasteurella multocida from septicemic cases in ostrich. These findings also supported by Rabab (2003) and Moursi and Husien (2007) serological serotypes of  Pasteurella multocida was found to be belonged to (5: A, 8: A, 9: A, 2: D and 12 untyped) (16.7%, 30.95%, 14.3%, 9.5% and 28.6) respectively. These serotypes are similar to those reported by Nasef et al (2003) and Fatma and Hala (2008) the sensitivity test of Pasteurella multocida serotypes revealed that ciprofloxacin, trimethoprim, Enrof loxaein, colistin suplate, Doxycyline, Amoxicillin chloramphenicol and oxyteteracycline. These results agreed with Zahraei and Farashi (2006) and Moursi and Husien (2007) and Fatma and Hala (2008). Such variation in sensitivity might be due to repeated use of special type of antibiotic therapy Jaslow et al. (1981), presence of resistant mutants strains, difference in geographics areas and time of isolation, may reflect differences in pattern of drug use, are due to recent isolates were more resident to antimicrobial against the old isolates of Pasteurella multocida (Chang, 1982).

Pasteurella multocida isolates was highly pathogenic for mice and chickens. The mortality in mice (70%, 80%, 60% and 50%) when infected by 5: A, 8: A, 9: A and 2: D) respectively but in chickens (90%, 80%, 60% and 60%) when infected by 5: A, 8: A, 9: A and 2: D respectively. In our study this agreed with Nasef, et al. (2003); Fatma and Hala (2008); Kapentonov, et al. (2000) and Engestrom et al. (2001) Table (4) and Table (5).

The liver sections showed vacuolar degeneration of hepatocytes with nuclear pyknosis and mononuclear cells infiltration around the blood vessels as well as perivascular edema these results agreed with Sohair and Moursi (2003); Hala and Kawther (2006) and Fatma and Hala (2008).

The heart lesions revealed edema among the degenerated and necrosed cardiac muscle fibers. The kidney revealed mononuclear cells infiltrations and edema in between the renal lobules. These results agreed with Amal and Mohamed (2000), Dunber et al. (2000), Muhoirwa (2000), El. Demerdach et al. (2003) and Refat and Fatma (2006) where they recorded that, these lesions may be attributed to the infection with bacterial pathogens specially Pasteurella multocida as well as vascular damage due then circulating toxins of E. coli, and Pseudomonas aeuroginosa as well as the kidney revealed mono nuclear cells infiltration and edema between the renal lobules. These results mainly similar to those reported by El-Demerdech et al (2003). In the present study, the liver showed hemorrhages and haemosiderosis. They also showed diffused necrosis and severe congestion of the blood vessels the results agreed with Amal and Mohamed (2000), Kamel et al (2001), El- Demerdach et al. (2003); Hala and Kawther (2006) and Fatma and Hala (2008). There fore, a wide spread pathological changes in different organs were recorded. In few cases, the heart showed myocardial edema and necrosis of muscle layers of myocardium as well as serofibrinous to organized pericarditis. These results were nearly similar to that described by Fatma (1999); Kamel et al. (2001); El-Dermerdach et al. (2003); Sohair and Moursi (2003), Hala and Kowther (2006) and Fatma and Hala (2008).

The spleen showed necrosis in some areas, edema and lymphoid depletion, these results agreed with Mohamed (2000) and Mouchira et al (2001) who stated that lymphoid depletion and necrosis of white pulps in the spleen.

The lung showed hyperplasia and mucinous degeneration of the bronchial epithelium. The kidney showed lobular nephrosis and marked kidney coagulative necrosis. The results nearly similar to those reported by El- Demerdach et al (2003), Sohair and Moursi (2003) Hala and Kawther (2006) and Fatma and Hala(2008). So, Successful management and disinfection of farms must be carried with therapy to dominate the predisposing factor as much as possible.

Control of the ostrich farms should be supplied with different laboratories to do periodical examination of birds to sure that free from diseases. Inside each ostrich farm, the birds should be undergoing the supervision of veterinary medicine to reach for correct diagnosis to give  suitable medication. At present no vaccination measures are recommended, but high standard biosecurity are required to prevent introduction of infections into flocks (Tally and Shan, 1996).

It could be concluded that the most bacterial pathogens which detected in ostrich farms were Pasteurella multocida, So. Successful management and disinfection of farms must be carried with therapy to eliminate the predisposing factor as much as possible.

The results of this study suggest that low hygienic measures at the farms slaughter houses and storage levels may be act as a factor in Pasteurella multocida contamination to minimizing risk of Pasteurella multocida. Hygienic measures should be improved in the farm, slaughter houses during transportation, storage and marketing, also sure that food handlers are not Pasteurella multocida carriers, in addition to rodents, wild birds and bests in and around farms.

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Table 1: Percentage of isolation of P. maltocida from ostriches cases in different Governorates: