PREVALENCE OF PASTEURELLA MULTOCIDA IN WATERFOWL WHICH SUFFERING FROM RESPIRATORY DISORDER AND ITS SENSITIVITY TO SOME DIFFERENT ANTIBIOTICS

Table 6: In vitro sensitivity of Pasteurella multocida isolates (No.10) to different antimicrobial agents.

Discussion

Pasteurella sp. is normal flora of the respiratory and gastrointestinal tract of many species of domestic and wild birds so it has primary or secondary role in pneumonia of poultry and the virulence of pasteurella is related to the polysaccharide capsule that allow the organism to resist phagocytosis.(Bailey and Scott^, s., 1994)

The severity and incidence of P. multocida infections may vary considerably depending on several factors associated with the host including species and age of the infected birds, the environment and the bacterial strain. No single virulence factor has been associated with the observed variation in virulence among strains. (Christensen and  Bisgaard, 2000)

Diagnosis depends on isolation and identification of the causative bacterium, Pasteurella multocida. Presumptive diagnosis may be based on the occurrence of typical signs and lesions and/or on the microscopic demonstration of numerous bacteria in blood smears, or impression smears of tissues such as liver or spleen

Clinical signs and gross lesions of the examined ducks and geese showed:

Sick birds appear exhausted, drowsy, loss of appetite other birds have convulsions, swim in circles, loss of appetite,, lameness, swollen and edematous wattles and combs, difficult breathing, cyanosis,  watery yellowish or green diarrhea, ruffled feathers, mucous discharge from the mouth,Similar Symptoms reported by Friend. (1987), Takahashi et al. (1996) and Woo and Kim (2006) they also showed that similar signs.

The most prominent lesions were hemorrhages of various sizes are found on the heart, liver, gizzard, and intestines. The liver may enlarged and darkened in color; a small number of necrotic and inflammatory foci were also discovered on the liver surface, heart and spleen. The lower portions of the digestive tract commonly contain thickened yellowish fluid, mild ascites and firm material collection in joints. Similar symptoms were recorded by Friend. (1987) and Kwon and Kang (2003) they reported clinical sings of Fowl cholera in all examined birds, including multifocal necrotic foci in the liver with enlargement, petechial hemorrhages on the heart, and mucoid exudates in the duodenal mucosa. Microscopically, there were hepatocytic necrosis with bacterial colonization, hemorrhage and necrosis in the myocardium, and hemorrhagic enteritis. Also Woo and Kim (2006) reported similar symptoms included orofacial edema, wattle and comb swelling, and respiratory symptoms. The cardiac air sac was filled with inflammatory materials, and serious pneumonic lesions were also found at the end of the lung lobe

Microscopical examination of blood samples and tissue smears of organs revealed bipolar staining bacillus and the pure colonies on blood agar plates showed transparent, glossy, and big colonies gave off a characteristic and sweet smell, Colonies range from 1 to 3 mm in diameter after 18-24 hours of incubation and are discrete, circular, convex,,translucent which is characteristic of P. multocida. Results of biochemical test of pure isolate illustrated on Table (3). These results agree with those reported by (Bisgaard et al., 1991; Murray et al., 1995; and Woo and Kim, 2006). They identification of Pasteurella species included Gram staining, motility test, glucose fermentation test, oxidase and catalase reactions. The strains obtained were typed by using the following reactions; ornithine decarboxylation, urease production, indole formation and production of acid from sucrose, maltose, mannitol, dulcitol, and sorbitol.

Bacteriological examination of samples according to the morphological characters, biochemical reactions of the recovered Pasteurella multocida were Tabulated in Table (1) revealed 134 (43.2%) out of 310 samples were positive to P. multocida. The positive samples included, 128 (51.2%) out of 250 waterfowl, 4 (13.3%) out of 30 water samples and 2 (6.7%) out of 30 Sediment samples.

Wetlands have long been suspected to be an important reservoir for Pasteurella multocida and therefore the likely source of avian cholera outbreaks P. multocida has been isolatedfrom the water and sediment of wetlands experience avian choleraepizootics and the bacteria can persist inwetland soil and water (Price and Brand 1984; Backstrand and Botzler, 1986)

 The incidence of P. multocida in both water and Sediments samples in our study were (13.3%) and (6.7%) respectively. These results are higher than that obtained by Samuel et al. (2004) They attempted to isolate P. multocida from 440 water and 440 sediment samples collected from 10 wetlands during winter and spring seasons. They could not be isolate P. multocida from envy samples. In contrast, they could isolated P. multocida from 20 of 44 wetlands, including 7% of the water and 4.5% of the sediment samples on the other side Lehr et al. (2005) could not be isolated P. multocidafrom 786 water and 786 sediment samples during winter 1997, while during winter1998 could be isolated  P. multocida serotype 1 from three enzootic and three reference wetlands at percentage of 14 out of 396 (3.5%) water samples and one out of 396 (0.3%) sedimentsamples .

Table (2) revealed 128 P. multocida isolates from all waterfowl samples (ducks and geese) with allover incidence of (51.2%). Out of these 92 (61.2%) from ducks and 36 (36%) from geese examined samples. We could be isolated P. mulotcida from apparent healthy, diseased, and freshly dead ducks at percentages of (10.0%). (66.7%) and (75.0%) respectively while from geese were (5.0%), (30.0%) and (66.7) respectively.

Our results are higher than that obtained by Nakamine et al. (1992) they isolated Pasteurella multocida from Fifty (25%) of 200 Muscovy ducks during outbreak of fowl cholera in Okinawa Prefecture of Japan,  Samuel et al. (1997) isolated P. multocida serotype 1 from an adult male goose and P. multocida serotype 3 from an adult female goose out of 298 lesser snow geese and also higher than recorded by  (Muhairwa et al., 1999) they detected of P. multocida in ducks at percentage of  (7%) in all three zones investigated, Mbuthia et al. (2007) they could be isolated P. multocida from only four chickens out of 123 birds examined, while could not be isolated from 24 ducks examined on the basis of biochemical characterization. Samuel et al., (2003) could be isolated P. multocida from apparently healthy geese. Also Lehr et al. (2005) found that a small proportion of 322 wild birds (<5%) were carriers of pathogenic P. multocida. On the basis of serology, an additional group of these birds (<10%) were survivors of recent avian cholera infection. They also found these birds were carriers of P. multocida even in the absence of disease outbreaks while, our results are lower than that obtained by Takahashi et al. (1996) they isolated Nine strains of Pasteurella multocida from Muscovy ducks, 5 to 6 dead ducks at percentage of (83.3%) and 4 of 8 ducks with percentage of (50.0%) from ducks have clinical of subacute disease characterized by lameness, corneal turbidity, and depression, and  Leotta et al. (2006) they could be isolated Fifty-five P. multocida gallicida, type A:1 from Eighty-six dead birds and one from water samples

Pathogenicity in vivo on mice

Results for the virulence of 3 strains of P. multocida on 3 groups of mice (each group contain 5 mice) within 24-48 hours after inoculation, recorded 20% mortality. by strain isolated from apparent healthy, 80% mortality with strain isolated from diseased birds and 100% with strain isolated from freshly dead birds, while no death from control group Table (4).

From direct smear, specimens of heart blood from dead mice were observed through the microscopic examination, bipolar staining bacillus were observed in all the specimens. This shows that there is a difference in mortality rate between strains isolated from examined ducks and geese due to difference in virulence of the strains.   

Pathogenicity in ducks:

Results of experimental infection of the susceptible ducks, instullated in Table (5) that the P. mulotcida isolates were pathogenic on experimental ducks through oral administration and I/T inoculation with a variety of mortality rates. It showed that, the first group which inoculated with strain isolated from apparent healthy cases was revealed 0 to 20 % mortality, and the second group which, inoculated with strain isolated from diseased birds was ranged from 60 to 80%, while was      80 to 100% in the third group which inoculated with strain isolated from freshly dead birds, while no death recorded in control group.

The clinical symptoms, direct smear of heart blood from dead birds revealed gram-negative, bipolar staining bacillus and post-mortem pictures of dead birds, are similarly that showing in examined ducks and geese samples in our work and agree with those reported by the author Samuel et al. (2003) they found that virulenceof P. multocida isolates from wetlands and Pekin ducks, ranged from (0 to 100%). In contrast, the virulence of isolates collected duringthe winter of 1997 from other wetlands ranged from (67% to 100%) and agree with reported those by Wobeser (1997) whoreported that virulence among strainswithin the same P. multocida serotype may be different. Samuel et al. (1997) reported that pathogenicity of the serotype 1 isolate was confirmed by inoculation in Pekin ducks (Anas platyrhynchos). while; serotype            3 isolate was non-pathogenic in Pekin ducks and could be isolated from pathogenic P. multocida serotype 1 from apparently healthy wild snow geese.

Antimicrobial drugs susceptibility

The extensive use of antibiotics as growth promoters and prophylactic agents for disease control in veterinary medicine has undoubtedly been responsible for large numbers of bacteria that have become resistant to different antibiotics. In-vitro tests the proper antimicrobial agents against P. multocida isolates, illustrated in Table (6) five strains were tested against 10 antimicrobial drugs. These strains somehow acquired a significantly high level of resistance to 3 types of antimicrobial drugs: Chloramephenicol (100%), Neomycin (100%) and Streptomycin (80%), while highly sensitive to 4 types of antimicrobial drugs which included, Penicillin (100%), Lincomycin (80%), Oxytetracycline (80%) and Ampicillin (80%). Some of these results are agreed with those obtained by (Morishita et al., 1996) they reported that P. multocida isolates were susceptible to Penicillin G, Sulfisoxazole, Tetracycline, and Trimethoprim-sulfamethoxazole. While, Woo and Kim (2006) they found that P. multocida was of high level of resistance to     7 types of antimicrobial drugs: Kanamycin, Neomycin, Oxytetracycline, Tetracycline, Tobramycin, Doxycycline, and even Gentamicin.

Conclusion:

From the abovementioned results, it can be concluded that a variation in virulence and survival of P. multocidamight explain why avian cholera outbreaks were observed withoutdetecting pasteurellae in the environment. Virulence among strainswithin the same P. multocida serotype may vary.

Strict control on entry of feed bags, equipment and personnel is necessary to exclude infection with fowl cholera. As well as, rodents need to be controlled and free-flying birds excluded. Carcass collection to reduce bacterial contamination in wetlandsis the current method used to control avian cholera in waterfowl.Other tools to control avian cholera have been suggested, includingtreatment of water chemistry to reduce the survival of P.multocida (Friend, 1999).

References

Backstrand, J.M. and Botzler, R.G. (1986): Survival of Pasteurella multocida in soil and water in an area where avian cholera is enzootic. Journal of Wildlife Diseases 22: 257–259

Bailey and Scott^, S. (1994): Genus pasteurella In: Diagnostic Microbiology 9^th Edition P. 420-422 (part4)

Bergan, T. and Norris, J.R. (1978): Pasteurella multocida, Biochemical characteristics and serotypes, p.271-292. In Methods in microbiology, volume 10, Academic press, New York.

Bisgaard, M.; Houghton, S.B.; Mutters, R. and Stenzel, A. (1991): “Reclassification of German, British and Dutch isolates so-called Pasteurella multocida obtained from pneumonic calf lungs”, Veterinary Microbiology, 26,

Blanchong, J.A.; Samuel, M.D.; Goldberg, D.R.; Shadduck, D.J. and Lehr, M.A. (2006): Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks J. Wildl Dis.; 42(1):33-9

Bredy, J.P. and Botzier, R.G. (1989): The effects of six environmental variables on Pasteurella multocida populations in water. Journal of Wildlife Diseases 25: 232–239

Christensen, J.P. and Bisgaard, M. (2000): Fowl choleraRev Sci Tech.; 19(2):626-37.

Cruickshank, R.; Duguid, J.P.; Marmoni, B.P. and Swain, R.H. (1982): Medical Microbiology.12^th Ed., Churonill Livingestone Edinburg, London, UK

Derieux, W.T. (1978): Response of young chickens and turkeys to virulent and a virulent Pasteurella multocida administered by various route. Avian Diseases 22, 131-139

Friend, M. (1987): Field guide to wildlife diseases.U.S. Fish Wildl. Serv., Resour. Publ. 167. 225 pp.

Friend, M. (1999): Avian cholera. In Field manual of wildlife diseases: General Field procedures and diseases of birds, M. Friend and J. C. Franson (Eds.). Information and Technology Report 1999-001, Biological Resource Division, U.S. Geological Survey, Madison, Wisconsin, pp. 75–92.

Glison, J.R. (2003): "Pasteurella and other related bacterial infection" In: Diseases of poultry edited by Saif, Y.M.Barnes.H.J.; Glison, J.R; Fadly, A.M.; McDougald, L.R. and Swayne, D.E. p.661 11^THEd.

Koneman, E.; Allen, S.; Damsel, ver and Somme, S.H. (1988): “Colour Atlas and text book of Diagnostic Microbiology 2NDJ.B Lip. Co. London.

Kwon, Y.K. and Kang, M.I. (2003): Outbreak of Fowl Cholera in Baikal Teals in Korea Avian Diseases, Vol. 47, No. 4, pp. 1491-1495.

Lehr, M.A.; Botzler, R.G.; Samuel, M.D.; Shadduck, D.J. (2005): Associations between water quality, Pasteurella multocida, and avian cholera at Sacramento National Wildlife Refuge. J. Wildl Dis.; 41(2): 291-7.

Leotta, G.A.; Chinen, I.; Vigo, G.B.; Pecoraro, M. and Rivas, M. (2006): Outbreaks of avian cholera in HopeBay, Antarctica J Wildl Dis.; 42(2): 259-70

Mbuthia, P.G.; Njagi, L.W.; Nyaga, P.N.; Bebora, L.C.; Minga, U.M. and Mugera, G.M. (2005): Indigenous Ducks are Better Reservoirs of P. multocida than Indigenous Chickens
Kenya Veterinarian Vol. 29  pp. 104-106

Mbuthia, P.G.; Njagi, L.W.; Bebora, L.C.; Mugera, G.M.; Ngatia, T.A.; Munyua, W.K.; Nyaga, P.N.; Minga, U. and Olsen, J.E. (2007): Preliminary Findings on the Carrier Status of Pasteurella multocida in Farmed and Traded Healthy-appearing Scavenging Indigenous Chickens and Ducks in Kenya Kenya Veterinarian > Vol. 31, No. 1 (2007)

Morishita, T.Y.; Lowenstine, L.J.; Hirsh, D.C. and Brooks, D.L. (1996): Pasteurella multocida in raptors: prevalence and characterization. Avian Dis.; 40(4): 908-18.

Muhairwa, A.P.; Mtambo, M.M.A.; Christensen, J.P. and Bisgaard, M. (1999): “Occurrence of Pasteurella multocida and related species in village scavenging poultry and animals that are kept in contact” (Submitted for publication).

Murray, P.R.; Baron, E.J. and Pfaller, M.A. (1995): Manual of clinical microbiology, 6th ed., p.505-506, American Society of Microbiology press, Washington, D.C.

Nakamine, M.; Ohshiro, M.; Ameku, Y.; Ohshiro, K.; Keruma, T.; Sawada, T. and Ezaki, T. (1992): The first outbreak of fowl cholera in Muscovy ducks (Cairina moschata) in Japan J. Vet. Med Sci.; 54(6):1225-7

Oxoid Manual (1982): The Oxoid manual of culture media, ingredients and other laboratory services 5^th Ed. Oxoid Limit

Price, J.I. and Brand, C.J. (1984): Persistence of Pasteurella multocida in Nebraska wetlands under epizootic conditions. Journal of Wildlife Diseases 20: 90–94

Price, J.I.; LYandell, B.S. and Portter, W.P. (1992): Chemical ions affect survival of avian cholera organisms in pond water. The Journal of Wildlife Management 56: 274–278.

Quinn, P.J.; Carter, M.E.; Makey, B. and Carter, G.R. (1994): Clinical Veterinary Microbiology Wolf Publishing an imprint of Mosby-year book Europe Limited, Lynton House, 7-10 Tavistock Spuare, London.WC IH9LB, England

Richard, R.B. and Glisson, J.R. (1997): Fowl cholera" In Diseases of poultry Edited by Calnek, B.W.. John, H. Barnes, Beard C.W. Medougald, L.R. and Saif, Y.M. p 143-159 10^th Ed.

Richard, W.V.; Harold, C.M. and Harold, H.B. (1967):  Fowl Cholera in waterfowl at squaw creek National Wildife Refuge, Missour. The Journal of Wildlife Management, Vol. 31, No. 2 pp.248-253.

Rimler, R.B. and Glisson, J.R. (1997): Fowl cholera. In Diseases of poultry, 10th Edition. B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. Mc- Dougald, and Y. M. Saif (eds.). The IowaStateUniversity Press, Ames, Iowa, pp. 143–159.

Samuel, M.D.; Shadduck, D.J. and Goldberg, D.R. (2004): Are wetlands the reservoir for avian cholera? J. Wildl Dis.; 40(3): 377-82

Samuel, M.D.; Shadduck, D.J.; Goldberg, D.R.; Wilson, M.A.; Joly, D.O. and Lehr, M.A. (2003): Characterization of Pasteurella multocida isolated from wetland ecosystems during 1996 to 1999. Journal of Wildlife Diseases 39: 798–807

 Samuel, M.D. Goldberg, D.R.;  Shadduck, D.J.; Price, J.I. and Cooch, E.G. (1997): Pasteurella multocida serotype 1 isolated from a lesser snow goose: Evidence of a carrier state. Journal of Wildlife Diseases 33: 332–335.

Stout, I.J. and Cornwall, G.W. (1976): Non-hunting mortality of fledged North American waterfowl. The Journal of Wildlife Management 40: 681–693.

Takahashi, S.; Sato, H.; Yamada, T.; Takenouchi, T.; Sawada, T.; Nakano, K. and Saito, H. (1996): Outbreaks of fowl cholera in Muscovy ducks (Cairina moschata) on a farm in Aomori Prefecture J. Vet. Med. Sci.; 58(3):269-72

Titche, A. (1979): Avian cholera in California. Wildlife Management Branch Administrative Report 79-2. California Department of Fish and Game, Sacramento, California, 49 pp

Wobeser, G.A. (1997): Diseases of wild waterfowl. Plenum Press, New York, New York, pp. 57–69

Woo, Y-Ku and Kim, J.H. (2006): Fowl Cholera Outbreak in Domestic Poultry and Epidemiological Properties of Pasteurella multocida Isolate the Journal of Microbiology, p. 344-353