SOME MICROBIOLOGICAL STUDIES ON PASTEURELLA MULTOCIDA IN RABBITS

Assiut University web-site: www.aun.edu.eg

SOME MICROBIOLOGICAL STUDIES ON PASTEURELLA MULTOCIDA IN RABBITS

EMAN H. MAHROUS ¹; M.W. ABD AL –AZEEM ²; S.A. AHMED ² and F.A. WASEL ¹

¹ Microbiology Department, Animal Health Research Institute. (Sohag Branch).

² Microbiology Department Faculty of Veterinary Medicine South Valley University.

Received: 20 September 2016;       Accepted: 24October 2016

INTRODUCTION

Pasteurella multocida is a gram-negative, non-spore forming, non-motile and capsulated coccobacillus that is penicillin-sensitive and belongs to the family pasteurellacae (Kuhnert and Christensen, 2008). Pasteurellosis or snuffles in rabbits is one of the most serious diseases which causes considerable economic losses in rabbit production units (Stelian et al., 2011). It is caused by pasteurella multocida which may cause death in rabbits or local infection as (rhinitis, otitis media and abscess), pneumonia and septicemia (Deeb et al., 1990). Infection by Pasteurella multocida can also appear without any clinical signs manifested (Jaglic et al., 2006).

The species of Pasteurella multocida is subdivided into four subspecies that include the type strain, multocida and three others, gallicides, septic and the recently described tigres (Petersen et al., 2001). It has 5 strains i.e. A, B, D, E and F and 16 serotypes (1 -16) (Azmat et al., 2013). Pasteurellosis in rabbits is mainly caused by the capsular type A and,  to a  lesser

Corresponding author: Dr. EMAN H. MAHROUS

E-mail address: dryasserkoubisy@gmail.com

Present address: Microbiology Department, Animal Health Research Institute. (Sohag Branch)

extent, capsular type D strains (Vandyck et al., 1995; Dabo et al., 1999). Diagnosis of the bacterium was traditionally based on clinical findings, culture and serological testing. Pasteurella multocida when grow in various media, yielded moderate to dense growth. Good growth is obtained in Brain heart infusion (BHI) and Casein sucrose yeast (CSY) while poor growth is obtained in Nutrient broth (NB) media (Mahmood, 2001). It will not grow on MacConkey agar and the colony growth is accompanied by a characteristic "mousy" odour that is due to metabolic products. On Leishman’s staining, heart blood smears and tissue impression smears prepared from liver, spleen and lung revealed characteristic bipolar organisms suggestive of Pasteurella multocida (Purushothaman et al., 2008).

Pasteurella species have both an oxidative and fermentative metabolism. Glucose and other carbohydrates are catabolised with the production of acid but without gas. Most species are catalase-positive and oxidase-positive; nitrates are reduced to nitrites by almost all species (Health Protection Agency, 2007).

Pasteurella multocida ELISA test was used for rapid serological identification of such pathogen according to Weber et al. (1993).As serology can't differentiate between current infection and previous exposure, the quickest and most accurate method for confirming an active Pasteurella multocida infection is molecular detection using polymerase chain reaction (Miflin and Balckall, 2001).

The aim of this work was carried out to isolation and identification of Pasteurella multocida from both apparently healthy rabbits and diseased ones, serotyping of the isolated microorganisms and characterization of pasteurella strain by (PCR).

MATERIALS AND METHODS

1. Animals and sampling:

A total number of 120 apparently healthy and diseased rabbits of different ages (60 of apparently healthy and 60 diseased rabbits) were obtained from three different rabbit farms at Sohag governorate. Samples were collected aseptically from blood and nasal swabs from rabbits in sterile packet and transported to laboratory in an ice box for bacteriological examination as soon as possible.

2. Isolation and identification of Pasteurella multocida:

The prepared samples were inoculated under aseptic conditions in enriched nutrient broth incubated for 20-24h at 37ᵒC.nasal swabs samples and a loopful from each blood samples were streaked onto blood agar and casein/sucrose/yeast (CSY) agar containing 5% sheep blood. Suspected colonies were selected and subcultured onto 5% sheep blood agar, CSY and MacConkey then checked for suspected Pasteurella multocida colonies. Blood film from heart blood was stained by Leishman stain examined for their bipolarity of Pasteurella multocida. Suggestive colonies of Pasteurella multocida were subjected to biochemical identification Manasa et al. (2013).

3. Serological identification of Pasteurella multocida by ELISA:

The commercial Pasteurella multocida ELISA test kits (Glory Science Co., Ltd, China Manufacturers, China) were used for rapid serological identification of such pathogen according to Weber et al. (1993) then using indirect - haemagglutination test for capsular serotyping and agar gel precipitation test for somatic serotyping.

4. Pasteurella multocida specific polymerase chain reaction (PM-PCR):

PCR has been proved to be useful in the detection of DNA of Pasteurella multocida (Purushothaman et al., 2008; Townsend et al., 1998). The success of PCR depends on the method of DNA extraction. The addition of Guanidine thiocyanate, Cetyltrimethyl ammonium bromide, Phenol extraction to the specimen is one of the options for DNAextraction. In the present study, simple boiling method was used to extract the DNA of Pasteurella multocida which makes the PCR technique an even more rapid and effective technique for Pasteurella multocida identification. Amplifying DNA fragment with in KMT1 gene using the Primers (KMT1SP6 and KMT1T7). In comparison with standard molecular weight marker (100bp), the molecular weight of the PCR products of all the isolates were found to be 460bp specific for Pasteurella multocida and confirmed the isolates as Pasteurella multocida.

RESULTS

Table 1: Observations of cultural and morphological characters of Pasteurella multocida.

Table 2: Biochemical activity of Pasteurella multocida isolated from apparently healthy rabbits and diseased ones.

Table 3: Biochemical characterization of Pasteurella multocida.

Table 4: Serological typing of Pasteurella multocida isolated from rabbits.

Fig. 1: showed Lane M: 100 bp ladder as molecular size DNA marker, Lane 1: Control positive Pasteurella multocida, Lane 2: Control negative DNA free sample and lanes 3 to 7: positive Pasteurella multocida and these isolates gave a single amplified product of the expected size of 460 bp.

Fig. 2: Showed Lane M: 100 bp ladder as molecular size DNA marker, Lane 1: Control positive Pasteurella multocida, Lane 2: Control negative DNA free sample and lanes 3 to 11: positive Pasteurella multocida.

Table 5: PCR confirmation of Pasteurella multocida isolates using species specific primers.

Table 6: Occurrence of Pasteurella multocida isolated from different samples of apparently healthy rabbits and diseased ones.

DISCUSSION

All isolates were subjected to full phenotypic characterization that showed in Table (1) revealing that all isolates were typical non-haemolytic, smooth, grayish glistening translucent and dew drop like colonies and their diameter was 1mm approximately on blood agar but these colonies were larger when grow on CSY agar. Direct smears from fresh samples that stained with gram's and leishmans stain's show gram negative capsulated cocco bacilli with bipolarity. These results are reported also by Abdullah et al. (2013); Bhimani et al. (2014).

Biochemical reactions of isolates as recorded in Table (2) revealed that (15) isolates out of all suspected isolates recovered from blood, nasal swabs samples of apparently healthy and diseased rabbits were positive  biochemically with apercentage of (28.3%). These results go hand in hand with Ekundayo et al. (2008); Manasa et al. (2013).

As well as these isolates were non motile and positive for oxidase, catalase and in dole test but negative for urea hydrolysis. Also able to ferment glucose, mannitol and can't ferment lactose. As shown in Table (3) these results go hand in hand with the previous results of Purushothaman et al. (2008); Abdullah et al. (2013) and Azmat et al. (2013).

The recorded date in Table (4) showed that 15 suspected organisms isolated from examined samples were submitted for ELISA test for Pasteurella multocida and revealed that Pasteurella multocida isolate from rabbits belong to capsular serotype (A) which agreed with the results ofJonas et al. (2001) and Tahmatan et al. (2014) and somatic serotypes belonged to 3:A (8 isolates) with a percentage of 53.3%, 12:A (4 isolates) with a percentage of 26.7%, 1:A (1 isolates) with apercentage of 6.7% while the remaining two strains ( 13.3%) were untypable. These results nearly coincided with those obtained by Okerman (1993).

The results illustrated, as in Table (5), that 14 out of 15 isolates were positive Pasteurella multocida with apercentage of 93.3% of the examined isolates. The specificity of the primer was confirmed by positive amplification of fragment with the extracted DNA of the bacterial isolates at expected size of 460 bp as shown in Fig. (1) and(2).

The obtained results in Table (6) proved that the occurrence of Pasteurella multocida of apparently healthy rabbits was 8.3%. These results nearly coincided with that reported by Eslam (2015), who found an isolation incidence of 9.42%, and Nada (1994), who examined 53 nasal swabs originated from apparently healthy rabbits, and isolated Pasteurella multocida with an incidence of 9.1%. In the present work, incidence of Pasteurella multocida among diseased rabbits was 15%. These results are nearly in accordance with Ibrahim (1993); Hussein (2000) and Hussein (2004) who isolated pasteurella multocida with an  incidence of (12%) , (12.3%) and (12.6%) respectively.

CONCLUSION

Under conditions of this investigation and according to data obtained, it was concluded that:-

1. PCR assay is highly sensitive and specific method for detection and identification of Pasteurella multocida than conventional biochemical and serological tests as a rapid technique.

2. Pasteurella multocida is one of the most serious pathogens of rabbits; it causes considerable economic losses in rabbits production units.

3. Rabbit's infection of Pasteurella multocida represent great harm to human health as the rabbits consider one of the most important source of food for humans.

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