Dear Dr. Santrac, About the problem you encountered, I didn't understand correctly if you have difficulty isolating Pasteurella spp. only from the lungs ... or chicken blood ... and you do not identify them on the culture plates ....it is correct?. What culture media do you use?. Have you tried isolating it from the trachea, liver or nasal sinuses?.

Dear Alessandro,

Thanks for the answer,

sorry for not precise question: I am interested to find what are the reasons I cannot isolate Pasteurella sp,. from poultry organs and tissue.

I use 5% sheep blood agar, 37 C for 48 h with aerobic and microaerophilic conditions and I am not able to cultivate materials from a carcase that showing pathological findings characteristic for avian pasteurellosis. I trayed add chicken erythrocytes in agar base, and that also does not give better isolation results. Is obvious that in lung and blood smears, and some other materials, from diseased animals and slides with Loeffler staining procedure made from them, I find bipolar microbe.

I thought maybe is better media or different conditions for growing Pasteurella from poultry.

It can be, that there are antibiotics in tissue and maybe that can be a reason for my failure to growth organism that Pasteur historically isolate under basic conditions more easily.

I can’t answer you, because i dont have domain.

Thank you .

Dear Violeta Santrač,

herein I am sending the protocols for the isolation and characterization of Pasteurella multocida from poultry.

Sincerely yours, Dr. Bratko Fililipič (email: [email protected])

------------------------------------------------------------------------------------------------------

Collection of Samples Samples were collected mainly from apparently healthy birds and mammals, cases of field outbreaks and from birds and mammals brought from farms for disease diagnosis and post mortem examination. The samples included Duck (n = 38), Fowl (n = 22), Deer (n = 4) and Turkey (n = 7). Isolation of Pasteurella multocida Samples of heart blood, lung, liver and spleen collected from the sacrificed/dead birds and mammals suspected to be suffering from pasteurellosis were streaked onto blood agar plates and incubated at 37 LC for 24–48 h in a candle jar. Heart blood smears and impression smears from liver, spleen and lungs were also prepared. Colonies that were suggestive of Pasteurella sp. were stained by Gram’s staining technique. The blood and tissue impression smears prepared were stained by Leishman’s staining technique and examined under oil immersion objective of the microscope for the presence of bipolar organisms. Phenotypic Characterization The isolates were checked for hemolysis on blood agar, growth on Mac Conkey agar, possession of capsule, Gram’s reaction and full biochemical characterization [8]. The tests included indole production, methyl red and voges proskauer reactions, urease activity, H2S production, nitrate reduction, citrate utilization, lysine and ornithine decarboxylase activities and production of acids from carbohydrates (glucose, galactose, inositol, lactose, malt-ose, mannitol, mannose, salicin, sucrose, dulcitol, sorbitol, trehalose, xylose and arabinose) [9]. The biotyping was done as per Mutters et al. [10]. Non Serological Capsular Typing Non serological capsular typing was done according to the method of Carter and Rundell [11]. Antibiogram Mueller–Hinton agar was used to study the antibiotic sensitivity pattern of the isolates. The antibiotic discs with known concentrations as noted in micrograms (lg) or nternational units (IU) per disc were used. Antibiotic sensitivity test was done as per standard single disc diffusion method of Bauer et al. [12]. Pathogenicity Testing of Isolates Swiss albino mice, 6–8 weeks of age were procured from the small animal breeding station (SABS), College of Veterinary and Animal Sciences, Mannuthy. Each mouse was inoculated intra-peritoneally with 0.1 ml of inoculum containing 3 9 108 organisms per ml in sterile normal saline. A control mouse was injected with 0.1 ml of sterile saline. All the mammals were observed for the clinical signs of infection. Blood smears were prepared from the dead mice and stained with Leishman’s stain. Re-isolation of P. multocida from heart blood, lung, liver and spleen of the dead mice were carried out. Results and Discussion Isolation of P. multocida was attempted from 155 samples originating from different birds the rate of isolation was 6.45 %. Ten isolates were obtained which consisted of 7 from ducks, 2 from fowl and 1 from deer (DP 36, DP 37, DP 38, DP 39, DP40, DP41, DP 42, FP 3, FP 4 and DeP3). The isolates produced small glistening dew drop like colonies on blood agar plates after incuba-tion at 37 LC for 18 h and appeared as Gram negative cocco-bacilli with Gram’s staining. There was no growth on Mac Conkeys agar and the bacteria were non motile and non hemolytic on blood agar. All isolates which produced indole were catalase and oxidase positive and reduced nitrate. There was no reaction with citrate, methyl red and voges-proskauer (Table 1). In the blood smear of infected poultry, bipolar organisms were detected (Fig. 1). All the isolates fermented glucose, galactose, mannose and sucrose, utilized ornithine decarboxylase and none utilized lactose. Fermentation of sugars like dulcitol, sorbitol, trehalose, xylose and ornithine decarboxylase by P. multocida isolates was studied to biotype the isolates (Table 2). Based on this, the isolates DP 39, DP 40 and FP 3 fermented sorbitol, trehalose and dulcitol and but did not ferment xylose. The isolates were biotyped as P. multocida sub sp. gallicida. All other seven isolates DP 36, DP 37, DP 38, DP 41, DP42, FP4 and DeP3 fermented trehalose, sorbitol and xylose but not dulcitol. All these isolates were biotyped as P. multocida sub sp. multocida. In non-sero-logical capsular typing the non-encapsulated isolates regained their capsule by mouse inoculation and were reisolated from the liver of mice (Fig. 2). All the 10 isolates were serotyped as CAP-A. In the antibiogram of P. multocida isolates, all of them were uniformly sensitive to nitrofurantoin and chloramphenicol (Table 3). The iso-lates DP36 and DP40 were resistant to gentamicin whereas all other isolates were sensitive to it. Only DP39 and DP40 were sensitive to sulphadiazene. All the isolates of P.multocida were able to kill the weaned mice by 24 h. The gross lesions observed in the internal organs of dead mice were petechiae in the pericardium, congestion of lung, liver and spleen. Heart blood smears and impression smears from spleen and liver collected from dead mice, stained with Leishman’s stain revealed the presence of bipolar organisms. P. multocida was re-isolated from the heart blood, lung, liver and spleen, on bovine blood agar. In the present study nine isolates were obtained from poultry and only one isolate was obtained from mammal (deer). Deer were among the first species of mammals in which pasteurellosis was originally described [13]. P. multocida septicemia has since been recorded sporadically in various species of deer in different countries [13]. Although P. multocida affect a wide variety of animals, strains isolated from non-avian hosts generally do not produce fowl cholera [14]. Strains that cause fowl cholera represent a number of immunotypes, which complicate widespread prevention by using bacterins. Chronically infected birds are considered to be a major source of infection [14]. P. multocida, a potentially pathogenic organism, which has been incriminated in both human and animal infections where it often causes severe respiratory abnormalities that can terminate in death [15]. Similar isolation work has been carried out from gut of Antarctic skua [16]. The successful isolation of P. multocida in this study is interesting not only because of its traditional role as a disease causing aerobic bacterium but also because of its toxicity, making it a highly invasive, pathogenic and virulent microorganism [17, 18]. Its ability to initiate infection in different anatomic structures of the body in human beings as well as in other animals has been reported to have deleterious effects on organs systems and immune responsiveness [17, 18]. Conclusion Fowl cholera and Haemorrhagic septicemia have gained importance as they cause huge economic loss both in poultry and livestock industry. In the present study the rate of isolation shows that the isolates were of capsular type A suggesting that it is the most common cause of fowl cholera. References 1. Marchart J, Dropmann G, Lechleitner S, Schlapp T, Wanner G, Szostak MP, Lubitz W (2003) Pasteurella multocida and Pas-teurella haemolytica-ghosts: new vaccine candidates. Vaccine 21:3988–3997 2. Kumar AA, Harbola PC, Rimler BR, Kumar PN (1996) Studies on Pasteurella multocida isolates of animal and avian origin from India. Indian J Comp Microbiol Immunol Infect Dis 17:120–124 3. Chawak MM, Verma KC, Kataria JM, Kumar AA (2000) Char-acterization of indigenous isolates of avian Pasteurella multo-cida. Indian J Comp Microbiol Immunol Infect Dis 21:111–114 4. Mulbagal AN, Kulkarni WB, Paranjape VL (1972) Some obser-vations on pasteurellosis in ducks. Indian Vet J 49:544–546 5. Rimler RB, Glisson JR (1997) Cholera. In: Calnek BW (ed) Dis-eases of poultry, 10th edn. Mosby-Wolfe, London, pp 143–159 6. Cowan ST, Steel KJ (1970) Manual for the identification of medical bacteria. Cambridge University Press, Cambridge 7. Cruickshank R, Duguid JP, Marmion BP, Swain RHA (1975) Medical microbiology vol 2, 2nd edn. Churchill Livingstone, Edinburgh 8. Blackall PJ, Pahoff JL, Marks D, Fegan N, Morrow CJ (1995) Characterization of Pasteurella multocida isolated from fowl cholera outbreaks on turkey farms. Aust Vet J 72:135–138 9. Barrow CI, Feltham RKA (1993) Cowan and Steel’s manual for identification of medical bacteria, 3rd edn. Cambridge University Press, Cambridge, p 331 10. Mutters RS, Ihm P, Pohl S, Frederiksen W, Mannheim W (1985) Reclassification of the genus Pasteurella trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis and Pasteurella langaa. Intl J Syst Bact 35:309–322 11. Carter GR, Rundell SW (1975) Identification of type A strains of Pasteurella multocida using staphylococcal hyaluronidase. Vet Rec 96:343 12. Bauer AW, Kirby WMM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496 13. Jones TO (1982) Outbreak of Pasteurella multocida septicaemia in fallow deer (Dama dama). Vet Rec 110:451–452 14.Ahir CL, Roy L, Jhala MK, Bhanderi BB, Mathakiya RA, Bhatt VD, Padiya KB, Jakhesara SJ, Koringa PG, Joshi CG (2011) Genome sequence of P. multocida subsp. gallicida Anand1_poul-try. J Bacteriol. doi:10.1128/JB.05706-11 15. Dritz SS, Chengappa MM, Nelssen JL, Tokaach MD, Goodband RD, Nietfeld JC, Staats JJ (1996) Growth and microbial flora of non medicated, segregated, early weaned pigs from a commercial swine operation. J Am Vet Med Assoc 208:711–715

16. Pramod WR, Kuddus et al (2007) Microbial flora of gut of Ant-arctic skua. Proc Natl Acad Sci India B 77:84–87 17. Hall MR, William PR, Rimler RE (1987) A toxin from Pasteurella multocida serogroup D enhances swine herpes virus 1. Replication and lethality in vitro and in vivo. Curr find Microbiol 15:277–281 Chiville NF, Rimler RH (1989) A protein toxin from Pasteurella type D causes acute and chronic hepatic toxicity in rats. Vet Pathol 26:146–147

Please check figures and tables in attachment " Untiteled"!!

Respected dr Filipic,

thanks for answer but seems that we well know that protocol.

We are good with Pasteurella isolation from mammalian species.

And that one from the first step does not work well under our condition.

as you add

* were streaked onto blood agar plates and incubated at 37 LC for 24–48 h in a candle jar.

is not working on poultry samples, with seep and/ or chicken blood in agar base. We newer use bovine or horse blood and for experiment we increased temperature on 42 C but that also did not make any difference.

We are looking for some other protocol that can work better on farm poultry tissue samples or other isolation procedures that can minimize unknown inhibitory factors.