Subscribe Now Subscribe Today
Abstract
Fulltext PDF
References
Research Article
 
Isolation and Identification of Mannheimia haemolytica and Pasteurella multocida in Sheep and Goats using Biochemical Tests and Random Amplified Polymorphic DNA (RAPD) Analysis



A.D. Hawari, D.S. Hassawi and M. Sweiss
 
ABSTRACT

The aims of this study were to isolate the M. haemolytica and P. multocida from sheep and goats of Jordan and to identify these bacteria by using biochemical tests and molecular technique. One hundred and ninety six samples were collected from nasal and throat of sheep and goats of two regions (Middle and Northern) in Jordan. The results of the biochemical tests and API 20 E kits identified 50 isolates of Pasteurella multocida and 5 isolates of Mannheimia haemolytica. The identification of these isolates was confirmed by polymerase chain reaction (PCR) technique using PMOut primers for P. multocida and PHSSA primers for M. haemolytica and by random amplified polymorphic DNA (RAPD) using the random primer (OPA-11). Fourteen RAPD profiles were found in P. multocida and 2 profiles were found in M. haemolyitca. The results of this study indicated that identification of P. multocida and M. haemolytica by RAPD analysis was in accordance with those of biochemical tests and using of more than one RAPD primers could improve the identification of the isolates.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

A.D. Hawari, D.S. Hassawi and M. Sweiss, 2008. Isolation and Identification of Mannheimia haemolytica and Pasteurella multocida in Sheep and Goats using Biochemical Tests and Random Amplified Polymorphic DNA (RAPD) Analysis. Journal of Biological Sciences, 8: 1251-1254.

DOI: 10.3923/jbs.2008.1251.1254

URL: https://scialert.net/abstract/?doi=jbs.2008.1251.1254

INTRODUCTION

Pasteurellosis broadly refers to any of the disease conditions caused by species of the genus Pasteurella (Dziva and Mohan, 2000; Davies et al., 2003). Pneumonic pasteurellosis is an acute infectious disease that causes widespread financial losses because of death, reduced live weight, delayed marketing, treatment costs and unthriftness among survivors (Aielo et al., 1998; Davies et al., 2001; Ozbey et al., 2004).

Mannheimia haemolytica, formerly known as Pasteurella haemolytica, (Tefera and Smola, 2002; Christensen et al., 2003; Sisay and Zerihun, 2003) is the bacterium most frequently isolated from shipping fever, which affects sheep and goats of all ages world wide (Falade, 2002; Ozbey et al., 2004). Mannheimia species naturally inhabit the upper respiratory system (tonsils and nasopharynx) of healthy sheep and goats and other wild and domestic animals (Chen et al., 2002).

Mannheimia haemolytica is the etiological agent of both bovine and ovine Pneumonic pasteurellosis (Dziva and Mohan, 2000; Davies et al., 2001; Falade, 2002), although various serotypes of Pasteurella multocida are occasionally involved. They are non motile, non spore forming, aerobic, fermentative, gram negative coccobacilli (Kodjo et al., 1999; Chen et al., 2002) which may show bipolar staining and may grow on most laboratory media with the exception of bile containing media such as MacConkey agar (Lariviere, 1992).

Infections result when an animal is compromised by any of a variety of stress factors as inclement weather, shipping (transportation), malnutrition, bacterial invasion of host defense, viral infections, nasopharyngeal colonization and dehydration, (Radostits et al., 1994; Baron, 1996; Aielo et al., 1998). Various M. haemolytica virulence factors influence the outcome of bacterial-host interactions (Davies et al., 1997).

Bacterial species included within the genera pasteurella and mannheimia have been classified on the basis of their phytogenetic characteristics (Tefera and Smola, 2002). Capsular serotyping provides the primary basis for the classification of strains and epidemiological typing of M. haemolytica (Peterson et al., 2001). Furthermore the purified organism is subsequently classified according to phenotypic traits such as morphology, carbohydrates fermentation patterns and serological properties. However, culture conditions can influence the expression of these attributes thus diminishing the stability and reliability for phenotypic methods for strain identification (Matsumoto and Strain, 1993). So, genotyping techniques have been used extensively to differentiate epidemiologically significant strains of P. multocida (Lainson et al., 2002).

Random amplified polymorphic DNA (RAPD) analyses has been applied for the distinction of strains belonging to the same species (Williams et al., 1990). It is a fast, sensitive method for the epidemiological studies and PCR-based method of genetic typing based on genomic polymorphisms. Ozbey et al. (2004) reported that some strains of Pasteurella multocida and Mannheimia haemolytica which isolated from cattle, sheep and goats were typed by using RAPD assay.

The aims of this study were to isolate the bacteria M. haemolytica and P. multocida from sheep and goats grown in different areas of Jordan and to confirm the identification of these bacteria by using biochemical reactions and Random Amplified Polymorphic DNA (RAPD) technique.

MATERIALS AND METHODS

Samples collection: Nasal and throat swabs (196 samples) from sheep and goats were collected randomly from different locations in Jordan during 2006-2007; pneumonic lungs and tonsils from slaughter house were also collected. The swabs were placed in sterile tests tubes containing 2 mL of transport medium (Amies medium) and kept immediately in an ice box for further analysis. The samples were then cultured overnight at 37 °C in Erlenmeyer flasks containing 200 mL of brain heart infusion broth.

Bacterial isolation and identification: Following incubation, samples from each culture were plated on blood agar selective medium for P. multocida and M. haemolytic (Oxoid) and on MacConkey agar. The inoculated plates were incubated aerobically and anaerobically at 37 °C for 24-72 h. Following purification, through subculturing, the isolates were subjected to further identification. For the laboratory detection of P. multocida and M. haemolytica, the identification of suspected bacteria colonies was achieved by observation of colonial morphology under microscopy and the use of some biochemical tests: hemolysis, motility, indole formation, litmus milk, glucose, saccharose, lactose, oxidase and catalase. Assay for biochemical properties of the bacteria isolates were conducted according to MacFaddin`s method (MacFaddin, 2000). For reliable identification and comparison of results, the API 20 E system (Biomereiux, France) was used. The bacteria were stored at -70 °C in 50% glycerol in brain heart infusion broth for further use.

DNA Extraction and Polymerase Chain Reaction (PCR): DNA extraction was carried out as previously described by Ozbey et al. (2004) with minor modifications. Identification of P. multocida and M. haemolytica were confirmed by PCR using PMOut primers (5`- AGG TGA AAG AGG TTA TG-3` and 5`- TAC CTA ACT CAA CCA AC-3`) for P. multocida and PHSSA primers (5`-TTC ACA TCT TCA TCC TC-3` and 5`- TTT TCA TCC TCT TCG TC-3`) for M. haemolytica derived from Omp87 and ssa gene, respectively (Ozbey et al., 2004). The PCR reaction was preformed in a total volume 25 μL containing 1X Go Taq reaction buffer, 2.5 mM MgCl2, 0.4 μM from each primer, 0.2 mM dNTPs (Bio Basic Inc., Canada), 1.25 U from Go Taq DNA polymerase (Promega Corporation, Madison, WI., USA) 1 μL from the isolated DNA.

The DNA was amplified under the following conditions in a PTC200 type thermocycler (MJ Research Inc., USA): denaturation step at 94 °C for 2 min, 40 cycles using the following settings: denaturation at 94 °C for 45 sec, annealing at 45 °C for 45 sec and extension at 72 °C for 1 min, followed by 5 min at 72 °C. The PCR product was analyzed on 1.5% agarose gel stained with 0.5 μg mL-1 ethidium bromide. Two DNA ladders 1 kb and 100 bp (Bio Basic Inc., Canada) were used to determine the size of the amplified fragments.

Randomly amplified DNA polymorphism: RAPD reaction was preformed as previously described by Ozbey et al. (2004) in a PTC200 type thermocycler (MJ Research Inc., USA). A random OPA-11 primer (5`-CA AT CG CC GT-3`) (Alpha DNA, Quebec) was used to determine the genetic differences among P. multocida and M. haemolytica isolates. Five microliter of amplified PCR products were analyzed on 1.5% agarose gel that stained with 0.5 μg mL-1 ethidium bromide and visualized under the UV light transilluminator. DNA molecular weight markers 1 kb and 100 bp DNA ladders (Bio Basic Inc, Canada) were used to determine the size of the amplified fragments.

RESULTS AND DISCUSSION

Gram stained of the respiratory and pneumonic lungs swabs yielded gram-negative coccobacilli. Direct leishmann`s staining of respiratory and pneumonic lungs smears revealed the presence of bipolarity, which is characteristic of Pasteurella sp. The results of the biochemical tests (Table 1), which conducted on suspected pure colonies, identified P. multocida and M. haemolytica. These results were confirmed by using API 20 E kit; all isolates presumed to belong to P. multocida were produced catalase, indol, presence of ornithine decarboxylase that fermented-mannitol, acid by fermentation of glucose and did not grow in MacConkey agar, while all isolates presumed to belong to M. haemolytica did not produce indole and grew in MacConkey agar. These results are in agreement with the findings by Tefera and Smola (2002).

Table 1: Results of biochemical reactions characteristics for M. haemolytica and P. multocida
+: Present, -: Not present

Table 2: Samples, isolates number and the site of isolation of M. haemolytica and P. multocida

A total of 55 isolates were identified out of; fifty isolates were P. multocida and five isolates were M. haemolytica (Table 2). The isolates were obtained from sheep and goats grown in two regions of Jordan, middle (42 isolates) and northern (13 isolates). On the basis of these results, P. multocida are the most common in sheep and goats in the middle region of Jordan. The serotypes of the isolates were not determined due to lack of serotypes in our laboratories.

The identification of P. multocida and M. haemolytica was confirmed by PCR technique. Amplification of genomic DNA from the two bacteria was conducted by using PMOut primers for P. multocida and PHSSA primers for M. haemolytica, corresponding to the anticipated sizes of 219 and 325 bp, respectively (Fig. 1). All P. multocida and M. haemolytica isolates that were positive by biochemical reactions were also detected to be positive by PCR. No amplifications were produced from the negative control. These results were in agreement with Ozbey and Muz (2006), who mentioned that All P. multocida chicken isolates that were positive by culture were also detected to be positive by PCR using PMOut 1-2 primers.

The profiles generated by RAPD analysis using OPA-11 primer with P. multocida and M. haemolytica isolates are shown in Fig. 2. Sixteen different profiles were found in this study; 14 profiles were found in P. multocida and 2 profiles were found in M. haemolyitca. The results of the RAPD assay indicated that more genetic heterogeneity exists among P. multocida and little genetic heterogeneity exists among M. haemolytica isolates. The results of M. haemolytica were in agreement with Ozbey et al. (2004), who indicated that little genetic heterogeneity exists among M. haemolytica isolates from cattle and sheep. Dziva et al. (2001) identified 81 P. multocida isolates of animal origin by both capsular typing and RAPD analysis; nine different strains with related RAPD profiles were observed. These results indicated that there was relationship between phenotypes and RAPD profiles.

Fig. 1: PCR product for 16 samples of Pasteurella multocida and Mannheimia haemolyitca where: Lane P1-P14 are Pasteurella multocida, P16 and P17 are Mannheimia haemolyitca; P15 and P18 are negative control, M1 and M2 represent 100 bp DNA ladder and 1 kb DNA ladder, respectively

Fig. 2: RAPD patterns for 16 samples of Pasteurella multocida and Mannheimia haemolyitca where: lane P1-P14 are Pasteurella multocida and P15-P16 are Mannheimia haemolyitca; M1 and M2 represent 100 bp DNA ladder and 1 kb DNA ladder, respectively

The findings of this study confirmed the specificity of primers PMOut and PHSSA for P. multocida and M. haemolytica, respectively and indicated that using OPA-11 in RAPD analysis is not efficient in differentiating isolates of M. haemolytica. It also indicated that molecular typing methods can provide a stable and highly useful analysis of bacterial isolates and have proved to be beneficial in reducing the limitations of the biochemical tests. In conclusion, the use of more than one RAPD primers could improve the identification of isolates by RAPD technique.

ACKNOWLEDGMENTS

We thank Al-Balqa Applied University for supporting this research and we thank Jordan Bio-Industries Center for technical support.

REFERENCES
Aielo, S.E., 1998. The Merck Veterinary Manual. 8th Edn., Merck and Co. Inc., New Jersey, ISBN: 09-911910-50-6, pp: 2305.

Baron, S., 1996. Medical Microbiology. In: Pasteurella, Yersinia, Francisella, Baron, S. (Ed.). University of Texas Medical Branch, Galveston (TX), ISBN:0-9631172-1-1.

Chen, H.I., K. Hulten and J.E. Clarridge, 2002. Taxonomic subgroups of Pasteurella multocida correlate with clinical presentation J. Clin. Microbiol., 40: 3438-3441.
Direct Link  |  

Christensen, H., M. Bisgard, J. Larsen and J.E. Olsen, 2003. PCR-detection of Hemophilus paragallinarum, Hemophilus somnus, Mannheimia (Pasteurella) haemolytica, Mannheimia spp., Pasteurella trehalosi and Pasteurella multocida. Methods Mol. Biol., 216: 257-274.
Direct Link  |  

Davies, L., R. MacCorquodale, S. Baillie and B. Caffrey, 2003. Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis. J. Med. Microbiol., 52: 59-67.
PubMed  |  Direct Link  |  

Davies, R.L., S. Arkinsaw and R.K. Selander, 1997. Evolutionary genetics of Pasteurella haemolytica isolates recovered from cattle and sheep. Am. Soc. Microbiol., 65: 3585-3593.
Direct Link  |  

Davies, R.L., T.S. Whittam and R.K. Selander, 2001. Sequence diversity and molecular evolution of the leukotoxin (lkt A) gene in bovine and ovine strains of Mannheimia haemolytica. J. Bacteriol., 183: 1394-1404.
CrossRef  |  

Dziva, F. and K. Mohan, 2000. Pasteurellosis and Pasteurella in Zimbabwe: An update. Zimbabwe Vet. J., 31: 1-10.
Direct Link  |  

Dziva, F., H. Christensen, J.E. Olsen and K. Mohan, 2001. Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida. Vet. Microbiol., 82: 361-372.
CrossRef  |  

Falade, S., 2002. Further Pasteurellaisolates from the republic of Zambia: A brief report. Trop. Vet., 20: 130-131.
Direct Link  |  

Kodjo, A., L. Villard, C. Bizet, J.L. Martel, R. Sanchis, E. Borges, D. Gauthier, F. Maurinand and Y. Richard, 1999. Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains. J. Clin. Microbiol., 37: 380-385.
Direct Link  |  

Lainson, F.A., K.D. Aitchison, W. Donachie and J.R. Thomson, 2002. Typing of Pasteurella multocida isolated from pigs with and without porcine dermatitis and nephropathy syndrome. J. Clin. Microbiol., 40: 588-593.
CrossRef  |  

Lariviere, S., L. Leblanc, K.R. Mittal and G.P. Martineau, 1992. Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis. J. Clin. Microbiol., 30: 1398-1401.
Direct Link  |  

MacFaddin, J.F., 2000. Biochemical Tests for Identification of Medical Bacteria. 3rd Edn., Williams and Wilkins, Lippincott, New York, ISBN: 0-683-05318-3.

Matsumoto, M. and J. G. Strain, 1993. Pathogenicity of Pasteurella multocida: Its variable nature demonstrated by in vivo passages. J. Avian Dis., 37: 781-785.
Direct Link  |  

Ozbey, G. and A. Muz, 2006. Isolation of aerobic bacteria from the lungs of chickens showing respiratory disorders and confirmation of Pasteurella multocida by polymerase chain reaction (PCR). Veterinarski Arhiv, 76: 217-225.
Direct Link  |  

Ozbey, G., A. Kilic, H.B. Ertas and A. Muz, 2004. Random amplified polymorphic DNA (RAPD) analysis of Pasteurella multocida and Mannheimia haemolytica strains isolated from cattle, sheep and goats. Vet. Med. Czech, 49: 65-69.
Direct Link  |  

Peterson, K. D., H. Christensen, M. Bisgaard and J.E. Olsen, 2001. Genetic diversity of Pasteurella multocida fowl cholera isolates as demonstrated by ribotyping and 16S rRNA and partial atpD sequence comparisons. Microbiology, 147: 2739-2748.
Direct Link  |  

Radostits, O.M., D.C. Blood and C.C. Gay, 1994. Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. 8th Edn., B. Saunders Co. Ltd., London pp: 1195-1199.

Sisay, T. and A. Zerihun, 2003. Diversity of Mannheimia haemolytica and Pasteurella trehalosi serotypes from apparently healthy sheep and abattoir specimens in the highlands of Wollo, North East Ethiopia. Vet. Res. Commun., 27: 3-14.
CrossRef  |  

Tefera, G. and J. Smola, 2002. The utility of ENTERORapid 24 Kit for the identification of P. multocida and M. haemolytica. Vet. Med., 47: 99-103.
Direct Link  |  

Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535.
CrossRef  |  PubMed  |  Direct Link  |  

©  2019 Science Alert. All Rights Reserved
Fulltext PDF References Abstract