Biochemical features of 123 Pasteurella /IIultocida strains isolated from chicken (94), ducks (22), ... Pasteurella multocida, a gram negative, nOill110tile, cocco-.
Indian JOllJ'llal ofAnimal Sciences 76 (6): 429-431, June 2006
Biochemical characterization of avian strains of Pasteurella I1lultocida in India S B SHIVACHANDRAI, A A KUMAR2, S JOSEPH3, M K SAXENA" VIJENDRA P SINGH' and S K SRIVASTAVtV'
Indian Veterinary Research Institute. Izatnagw; Uttar Pradesh 243 122 India Recdved: 3 February 1005; Accepted: I March 2006
ABSTRACT Biochemical features of 123 Pasteurella /IIultocida strains isolated from chicken (94), ducks (22), quails (4), turkey (2) and geese (l) belonging to different geographical regions ofindia were studied. All strains exhibited similar cultural, morphological and biochemical characteristics, however, they differed in their ability to utilize various sugar. Out of 123 strains, 7(5.69%),6(4.88%),89(72.36%), 70(56.9 I%),63(51.12%) and 14( 11.38%) strains decomposed arabinose, dulcitol, sorbitol, trehalose, xylose and lactose, respectively. A total of 12 distinct biovars (l to XII) were identified which categorized avian strains into a predominant P lIlultocida subsp. /IIultocida 1(67.48%), followed by P mu/tocida subsp. septica (26.02%), P l1lultocida subsp. gallidida (3.25%) and unrecognized biovars (3.25%). Key words: Avian, Biovars, Pasteurella lJlultocida
Pasteurella multocida, a gram negative, nOill110tile, coccobacillus, affects a wide range of avian species including wild and domestic animals, and is considered a highly variable pathogen due to its diversity observed among serotypes with respect to host predilection, pathogenicity, biochemical, cultural and antigenic specificity (Carter and Chengappa 1981 ). In India, the reports on biochemical characterization of P multocida strains are scanty (Kumar et al. 1969. Chawak et al. 2000). However, a few reports mentioning variability in sugar fermentation by avian P. mllltocida isolates have been reported (Rajini et al. 1995, Chawak et al. 2000). A variety of biochemical reactions have proven to be useful in characterization of P. l1211ltocida strains from avian origin. DNA hybridization studies (Mutters et al. 1985) revealed 3 homology groups of P. multocida that differ sufficiently from each other and they qualified as different species of Pasteurella. Ever since the recfassification of genus Pasteurella. there have been many studies I on the biochemical characterization of avian strains of P. multocida into 3 subsp. In view of the above, the present work was envisaged to characterize P 1l1ultocida strains biochemically and identify the prevalent subspecies among avians in India by using conventiona~_ methods to assess its applicability in
epidemiological studies of disease outbreaks.
Present address: 'Department of Biology, The Catholic University of America, Washington DC-200M, USA. 2'('Principal Scientist, 5Sen ior Scientist, 3Ph.D. Scholar, Division of Bacteriology and Mycology. 4Assistant Professor, Department of Veterinary Microbiology, Veterinary College, GB Pant University of Agriculture and Technology, Pantnagar 263 145.
Kumar et al. (1996) and Chawak et al. (2000) reported fowl cholera among avians. In the present study, 123 avian strains of P. multocida isolated fro111 cases of avian pasteurellosis belonging to different states of country exhibited similar cultural, morphological and biochemical characteristics as reported by previous workers and criteria
MATERIALS AND METHODS Source o/Pasteurella t11ultocida strains P l'Illiltocida strains (123) isolated from chicken (94), ducks (22), quails (4), turkey (2) and geese (1) died of pasteurellosis were obtained fro111 Pasteurella Repository, Division of Bacteriology and Mycology, IVRI, Izatnagar, India. Avian P lnultocida strains were previously obtained from different geographical regions ofIndia at difl'erent times (between 1991 to 2002) either as lyophilized cultures or recent isolation of blood agar slants and were further confirmed by conventional identification and scrotyping. Biochemical characterization Initially, all the received cultures were grown on BEl broth and further plated on blood agar plates to pick up a single colony for use in further characterization studies. Following the revival of P. multocida strains on blood agar, the cultural, morphological, biochemical and sugar fermentation tests were carried out by following standard procedures as per Cowan and Steel (1970) and Cruickshank et al, (1975).
RESULTS AND DISCUSSION
[Indian Journal ofAnimal Sciences 76 (6)
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unrecognized subsp. of P. nZllltocida (biovars VI and VII). The differentiating properties of each biovars and categorization of strains are given in Table 1 . The strains belonging to biovar I fenllented only sorbitol whereas the strains belonging to' biovar IV decomposed sorbitol and ,xylose, but did not ferment arabinose, trehalose and dulcito1. The biovars with the highest number ofstrains were I (18.7%) and IV (17.07%). The remaining 10 biovars with various fermentation variability accounted for rest of strains (65.23%). In contrast, 4 ofthe strains (2 fermented all sugars and 2 none) were not assigned to any of the 3 subspecies. Out of 123 strains, 83 (67.48%), 32 (26.02%) and 4 (3.25%) belonged to P multocida subsp. multocida, P ll'lultocida subsp. septica and P multocida subsp. gallicida, respectively, indicating that the majority of Indian avian strains of P multocida belong to P. rnultocida subsP. multocida. The predominance of P multocida subsp. multocida among avians was also noticed by Christensen et al. (1998) in Denmark, who examined phenotypic and genotypic characteristics of 89 P. multocida strains and assigned all the strains to P multocida subsp. multocida. The prevalence of P 111ultocida subps. gallicida in small number of strains (4) was also reported in this study. All 3 subspecies of P multocida have been isolated from cases offowl cholera, but the majority of strains reported from domestic as well as from wild birds have been identified as subsp. lnultocida (Snipes et at. 1990, Hirsh et al. 1990, Fegan et aI. 1995, Morishita et aI. 1996, Jurga and Opacka 2000). Among chicken strains (94), 60 (63.83%),26 (27.37%) and 4 (4.26%) strains belonged to P multocida subsp. multocida, P multocida subsp. septica, P. multocida subsp. gallicida, while 4 strains belonged to 2
enlisted in Bergey's Manual (Holt et al. 1994). Following revival ofstrains ofblood agar plates after incubation at 37°C for 18 h, exhibited similar cultural characteristics like small glistening mucoid dew drop colonies, which found to be Gram-negative cocco-bacilli when stained with Gram's stain. All strains produced indole, positive for catalase, oxidase, nitrate reduction and ornithine decarboxylase test. No reaction was seen with citrate, methyl red (MR) and VogesProskauer (VP) test, and gelatin liquefaction test. The organism did not grow on McConkey's agar and was found to be non-motile, non-haemolytic on blood agar. Glucose, fructose, galactose, mannitol, sucrose were fermented and none of the strains reacted with salicin, raffinose, inositol, and rhamnose. However, the strains differeq in their ability to ferment some sugar. Fourteen strains (11.38%) were lactose positive, indicating the fact that suc;h' variant, not numerically large, are prevalent in avians. The finding is similar to that of Heddlenston et al. (1976) who reported 1.6% of P multocida strains which were lactose positive out of 948 strains studied. In another study, 9 out of 150 straip.s appeared to be lactose positive valiants of P. multocida subsp. l1wltocida (Blackall et aJ. 1995). The current study noticed differences among strains in their ability to ferment arabinose, dulcitol, sorbitol, trehalose, xylose and lactose. These variabilities permitted us to classify straiils into 12 distinct biochemical types termed Biovars I to XII and then to include the strains into 3 subspecies of P multocida according to Mutters et al. (1985), which resulted in recognition of P. 11lultocida subsp. multocida (biovars I to V), P. multocida subsp. septica (biovars VII to X), P. multocida subsp. gallic ida (biovars XI and XII) and ~ab1e
1. Biochemical properties of avian P. multocida strains
---_... ---
_._-_._-
Fermentation of sugar
Biovars U
III
IV
V
VI
VII
VIII
L( +) arab inose + + + Dulcitol + Sorbitol + + + + + + Trehalose -+-+-++ D(-+-) xylose -+-++ + + Total 23 16 20 21 3 2 2 2 number of (13.01) (J 8.70) (16.26) (17.07) (2.44) (1.63) (1.63) (1.63) strains (%) rdentification militocida multocida multocida multocida multocida ? ? septica Chicken (94) (%) 15 10 20 2 13 I 2 2 (15.96) (10.64) (21.28) (13.83) (2.13) (2.13) (2.13) (1.06) Duck (22) (%) 4 6 1 7 (18,18) (27.27) (31.82) (4.55) Turkey (2) 1 Quail (4) 2 I Geese (1)
-----------_.-
IX
X
XI
XII
+ -+-
+ -I-
-I-
-+-
+ 14 (11.38)
16 (13.01)
septica 11
septica gallicida gal/ielda 14 1 3
( 11.70) 3
(14.89) 1
(I 3.64)
(4.55)
+ I (0.81 )
3 (2.43)
(1.06)
.....
~-----
(3.19)
...
_---
Number of strains positive for arabinose-7 (5.69%); dulcitol-6 (4.88%); sorbitol-89 (72.36%); trehalose-70 (56.91 %); xylose···63 (51.22%); lactos~-14 (11.38%). ?-Cannot be assigned to any recognized subspecies of P. multocida.
June 2006]
BIOCHEMICAL CHARACTERIZATION OF AVIAN STRAINS OF PASTEURELLA MULTOCIDA
biovars, which did not have felTIlentation patterns identical to any of the published P multocida subspecies. Our of 22 ducks strains, 18 (81.82%) and 4 (18.18%) belonged to P multocida subsp. multocida and P muItocida subsp. septica respectively. According to Hirsh et al. (1990) among 295 strains isolated from wild waterfowl, 62.7% of strains belonged to P multocida subsp. multoeida, 36.6% to P. lJlultocida subsp. gal1icida and 0.7% to P. multodda subsp. septiea. Although, it has been presumed that P. 112llltoeida subsp. gallicida is predominantly associated with webfooted birds (Christensen and Bisgaard 1997), we could not categorise any duck strain to P. mllltocida subsp. gallicida. Numerically, a few strains Oliginated from quail (4), turkey (2) and geese (1) were also grouped in to a major biovar P. mllltocida subsp. multocida and minor P. multocida subsp. septica. Turkey strains in the present study decomposed trehalose in addition to xylose and sorbitol, which were reported to be 100% in tune with the result observed by Walser and Davis (1975). Snipes et aZ. (1990) in USA also found Turkey strains belonging to all the 3 subsp. of P. multocida with predominant strains (95.9%) categorized as P multocida subsp. multocida. The present findings warrant further analysis ofmore number of strains from avian species before one could arrive at any conclusion. Although, avian strains of P. multocida are broadly classified into 3 subsp., a number of biovars based on variability in utilization of a individual sugars by certain strains were n01'lced. The variations in biovars observed in the current study, however, seems to be insignificant as they appear to confirm Mutters' original statement that variations in raffinose, lactose, maltose, trehalose, and D-xylose fermentation are of no taxonomic consequence (Mutters et al. 1985). Several variants within both P. multocida subsp. multocida and P. multocida subsp. septica were noticed in the literature (Blackall et af. 1995, Feganet aZ. 199,5, Blackall et al. 1997). Arabinose utilization by avian strains in India was lesser than 5.69%. The decomposition of arabinose varied (2 to 92%) among different avian hosts as per Heddleston (1976). The avian strains which utilize arabinose belonged to serotype A: 1, strongly suggesting the frequent observation made by earlier reports, however, it was interesting to note that these strains also decomposed xylose, which is in contrary to earlier reports. It was also accepted that the effect of P multocida strains on arabinose and xylose indicated the difference in their pathogenecity to birds and mammals; the strains pathogenic to birds ferment arabinose but not xylose whereas the strains pathogenic to mammals act inversely. A few strains (4.88%) decomposing dulcitol were found in the ~eUITent study, which was in contrast to the report of Dorsey (1963) who found an exceptionally high percentage (81.5%) among 432 avian strains, indicating changing pattern ofbiochemical properties ofavian strains under the influence of various incriminating stress factors in an intensive
431
commercial poultry farming. This presumption is supported by earlier examinations, which revealed that the percentages of the strains fermenting dulcitol ranged from 0 to 2 and with regard to sorbitol the percentage was 82. In this study, a high percentage ofthe strains decomposing sorbitol (72.36
[Indian Journal ofAnimal Sciences 76 (6)
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