Isolation , identification , characterization and antibiogram of Pasteurella multocida isolated from pigs in Mizoram with special reference to progressive atrophic rhinitis

Aim: Isolation and identification of Paturella multocida from pigs of Mizoram with or without clinical lesions of progressive atrophic rhinitis (PAR) followed by their detection, capsular typing and screening for toxA gene. Materials and Methods: Four hundred nasal swabs sampled from pigs were collected from 6 different districts of Mizoram. Swabs were processed for detection and isolataion of P. multocida by PCR and traditional bacteriological assays. Isolates were subjected for multiplex PCR for capsular typing, detection of toxA gene and characterization by RAPD-PCR. Isolates were also tested for antimicrobial sensitivity profile by disc-diffusion method. Results: A total of 21 swabs were found to be positive by P. multocida species specific PCR (PM-PCR) with an amplicon of 460 bp, of which P. multocida could be isolated from 15 swabs (3.75%). All the isolates were grouped under capsular type A (n=9) and D (n=6) by multiplex PCR. All the isolates were found to be negative for toxA gene by PCR, indicating the isolates as non-toxigenic. Isolates under similar capsular type provided a unique banding pattern by RAPD-PCR, whereas no discrimination was observed between serotypes. Conclusion: Although, no isolates exhibited toxA gene by PCR, isolataion of P. multocida of capsular type A and D may be an indication of their possible role in PAR in pigs of Mizoram.


Introduction
for induction of the experimental disease, development of improved diagnostics, development of effective Progressive atrophic rhinitis (PAR) is a widely therapeutic/ prophylactic pharmaceutical approaches prevalent, upper respiratory tract disease of swine and development of immunoprophylactic products for characterized by sneezing, snuffling, nasal discharge, effective control of the disease.

epistaxis, growth retardation, reduction in feed
There is paucity of information regarding PAR in utilization, degeneration and atrophy of the nasal pigs in India, particularly in North Eastern Region.turbinate bone leading to visible distortion and Therefore, the present study was conducted for shortening of the snout [1].Moreover, it has been isolation and identification of P. multocida from nasal recognized as a contributor to debilitating and fatal cavities of pigs of Mizoram with or without clinical porcine pneumonia for at least 120 years and continues lesions of PAR followed by their detection, capsular to be sustained, unabated with high prevalence in typing and screening for toxA gene by PCR based different part of the world [2].Toxigenic strains of P.
assays.multocida (both capsular type A and D) alone, or

combined with environmental factors like dust, ammonia and other physical or chemical irritants are
Sampling: 400 nasal swabs were collected from pigs of the primary causes of atrophic rhinitis [3].
different age groups of either sex from 6 districts of Livestock plays an important role in this hilly Mizoram during June 2010 to May 2011.Of the 400 state of Mizoram, as crop production is still traditional.pigs, 9 were showing respiratory distress as a suspected Amongst the livestock, pig is most important and case of atrophic rhinitis.Samples were collected from almost every family rears pig as backyard venture.
local household pigs as well as from organized pig Since the pig rearing is an important sector in rural farms.economy in this region, a better understanding of PAR 15 P. multocida were isolated and identified and with a high degree of discrimination between capsular designated as Pm1 to Pm15 (Table-1).P. multocida was type A and D. All 9 isolates of capsular type A showed isolated from all the 9 clinically suspected PAR pigs.similar banding pattern, and similarly, all 6 isolates of Out of 391 apparently healthy pigs, P. multocida was capsular type D exhibited same banding pattern (Fig - 3). isolated from only 6 pigs.
Antimicrobial sensitivity test: PAR in pigs can be Capsular typing of P. multocida by multiplex PCR treated with potentiated sulphonamides, ampicillin, assay: Of the 15 P. multocida isolates 9 (60%) and 6 tetracyclines, ceftiofur or enrofloxacin.Support (40%) were identified as capsular type A (1044 bp) and therapy such as additional food and electrolytes may be capsular type D (657 bp), respectively (Table-1, Fig- 2).
necessary.In the present study, majority of the isolates Of the 9 isolates from PAR suspected positive animals, exhibited 100% sensitivity against amoxicillin, 3 and 6 isolates were identified as capsular type A and cephalexin, chloramphenicol, ciprofloxacin, co-D, respectively.On the other hand, all the 6 isolates trimoxazole, enrofloxacin, erythromycin, gentamicin, from apparently healthy animals contained capsular ofloxacin, rifampicin and tetracycline.However, three type A only.
antibiotics did not show 100% sensitivity.The order of Detection of toxA gene by toxA gene specific PCR sensitivity were oxytetracycline (93%) followed by assay: All the 15 isolates tested for the toxA gene were ampicillin (87%) and amikacin (47%) (Table -3).found to be negative for an expected amplicon of 1230

bp.
The PM-PCR assay used in this study was highly

Randomly amplified polymorphic DNA -PCR (RAPD-
specific and sensitive and direct use of the confluent PCR): RAPD-PCR of all the 15 P. multocida isolates growth culture provided rapid detection of P. provided a range of amplicons between 400-1400 bp   multocida, regardless of the purity of the samples.
Toxigenic P. multocida is recognized as a Similar method of bacterial isolation was also followed causative agent of PAR in pigs.It has been isolated with by other research groups in India and abroad [8,9].In almost equal frequency from pigs with or without the routine diagnostic procedure, it is difficult to obtain a signs of PAR.However, the herds with clinical PAR pure culture of P. multocida from clinical samples had a higher prevalence of capsular type D toxigenic P. because of contaminants and/or death of organisms.
multocida and a lower prevalence of capsular type A Therefore, only PM-PCR positive samples could yield non-toxigenic P. multocida [8,14].Moreover, pigs with isolation of the organism.
signs of respiratory tract infection mainly developed All the 21 PM-PCR positive cultures in BHI broth the symptoms similar to pneumonia.The result of this were inoculated in mice individually for isolation of P.
study is also in accordance with the previously multocida.The organism was isolated from heart blood published reports by various research groups [9].and spleen of dead mice.When the same samples were Ranjan et al. [15] also described that toxA gene based inoculated directly on 5% sheep blood agar, isolation PCR can be used for direct analysis of toxigenic P. rate of P. multocida was poor (70%).Similar multocida without additional hybridization.This assay observations were also recorded by other workers with appears to be the most sensitive and effective method a success rate of approximately 18.0%, because of for large scale analysis of nasal and tonsillar swabs.frequent overgrowth of contaminants [10].Use of PM-RAPD-PCR showed that capsular type A and D P. PCR and subsequent mice inoculation for screening of multocida isolates exhibited two different banding nasal swabs drastically reduced the time for isolation of patterns ranging between 400-1400 bp (Fig - 3).The P. multocida.
correlation between RAPD pattern and capsular type The present study recorded relatively low helped in differentiating the isolates belonging to the recovery of P. multocida from nasal swabs collected same species.In the present study, it was also observed from Mizoram.This might be due to the random that RAPD pattern of P. multocida capsular type A was sampling of the animals.Although the sample size different from that of capsular type D, but no might be low for any conclusive remarks, it revalidates discriminatory patterns were recorded within the same the fact that P. multocida is a common inhabitant of pig capsular type (Fig. serotypes.The PCR based capsular typing assay [11] was Majority of the isolates were sensitive to amoxicillin, found to be a rapid and extremely reliable assay for cephalexin, chloramphenicol, ciprofloxacin, codetermining the capsular types of a large number of P. trimoxazole, enrofloxacin, erythromycin, gentamicin, multocida isolates.In the present study, 9 (60%) and 6 ofloxacin, rifampicin and tetracycline (100%) (40%) isolates were capsular type A and D, respecti-followed by oxytetracycline (93%), ampicillin (87%) vely.None of the isolates were untypeable.Capsular and amikacin (47%).The lower sensitivity of amikacin type F and untypeable are uncommon in pigs but are could be due to the gradual development of resistance often isolated from avian species.However, Choi et al.
enrofloxacin and ofloxacin along with chloramphemultocida isolates from pneumonic pigs in Korea.The nicol has also been reported by Sharma et al. [17] in an present study showed higher percentage of capsular in vitro study.Enrofloxacin and chloram-phenicol type A (60%), compared to capsular type D (40%), were found to be quite effective against pasteurellosis which is in agreement with previous studies [12,13].
by several research groups [18].against pasteurella, so as to find out an effective Singh, V.P. and Srivastava, S.K., (2004) Molecular antimicrobial agent to be used by the veterinarian.
variability among strains of Pasteurella multocida isolated from an outbreak of haemorrhagic septicemia in India.Vet.

typing of P. multocida isolates by multiplex extension at 72 C for 6 min. PCR assay: All the isolates were subjected to multiplex
PCR assay was conducted for the detection of P.
Detection of P. multocida by PCR assay: Each swab and its impact on animal husbandry and economy is was grown in Brain-Heart Infusion (BHI) broth essential.Studies on PAR should provide a foundation o overnight at 37 C.After incubation, 1 ml of bacterial suspension was centrifuged at 7084 g.The bacterial pellet was washed thrice with sterile normal saline solution (NSS) (0.85% w/v) and resuspended in 300 µl o supernatant was used as template DNA for PCR.PMfor 30 sec and a final extension at 72 C for 6 min.o Capsular o initial denaturation at 95 C for 5 min, followed by 30 Antimicrobial drugs sensitivity assay of P. multocida: o cycles of denaturation at 95 C for 30 sec, annealing at All the isolates were subjected to in vitro antibiotic Detection of toxA gene by toxA gene specific PCR animals, were found to be positive by PM-PCR for a assay: toxA gene specific PCR was performed [5] by a product of 460 bp, which is specific for P. multocida.colony touch PCR mixture (25 µl) containing 3.2 µM Of the 21 positive samples, 9, 3, 2, 2 and 5 were of each primer, 200µM of each dNTPs, 1x PCR buffer, sampled from Aizawl, Lunglei, Kolasib, Serchhip and 1.5 mM MgCl and 1 U of Taq DNA polymerase.The 2 Champhai district, respectively (Table-2 and Fig-1).following standard cycling procedure was used for o Isolation and identification of P. multocida: A total of DNA amplification: initial denaturation at 95 C for 5

Table - 1
. Oligonucleotide primers used in the present study

Table - 2
. PCR based screening and isolation of Pasteurella multocida from nasal swabs collected from 6 different districts of Mizoram.
3).Dutta et al. [6] observed the in the nasopharyngeal area.Isolation of P. multocida polymorphism in 33 isolates of P. multocida, isolated from aperantly healthy pigs indicated that these from rabbits and reported it as an efficient and reproduanimals were prone to disease under stress conditions.cible technique.Dutta et al. [6,16] characterized P. Kalorey et al. [11] have also recorded an outbreak of multocida strains of different serotypes by RAPD and pasteurellosis with high mortality in indigenous pigs in observed a unique banding pattern for individual India.

Table - 3
. Antimicrobial drug sensitivity assay of 15 P. multocida isolates , no isolates exhibited toxA gene by 5. Babita Devi L (2012) Molecular characterization of Pasteurella multocida of porcine origin.Ph.D. theis.PCR, isolataion of P. multocida of capsular type A and Submitted to Assam Agricultural University, Jorhat, Assam.D may be an indication of their possible role in PAR in 6. Dutta, T.K., Singh, V.P. and Kumar, A.A. (2009) Molecular pigs of Mizoram.In the last two decades, shift in detection and characterization of Indian isolates of antibiotic sensitivity spectrum of pasteurella is well Pasteurella multocida serogroup D. Indian.J. Anim.Sci.79: 11-14. Although