2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 165-171, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000021-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.21

Antibacterial Sensitivity and Detection of Virulence Associated

Gene of Pasteurella multocida Isolated from Rabbits

Fatma M. Mohamed¹*, Marium F. Mansy², Abd-El-Twab M. Abd-Al-Jwad³and Ahmed K.Hassan⁴

^1,3Poultry Diseases, Assiut Regional Laboratory, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

²Bacteriology, Assiut Regional Laboratory, Animal Health Research Institute, Agricultural Research Center (ARC), Egypt.

⁴Department of Poultry diseases, Faculty of Veterinary Medicine, Assiut University, Egypt.

^*Corresponding author’s Email: fmmi66@yahoo.com; ORCID: 0000-0003-3393-4638

Received: 02 Feb. 2020

Accepted: 12 Mar. 2020

ABSTRACT

The aim of the present work was to determine antibacterial sensitivity and resistance patterns of Pasteurella

multocida isolated from rabbits in different farms of Assiut Governorate. Also, this study aimed to detect virulence-

associated gene (toxA) of Pasteurella multocida. A total of 40 freshly dead rabbits were used to collect samples from

liver, lung and subcutaneous abscess. In addition, tracheal swab samples were collected from 20 diseased rabbits.

Bacteriological examination revealed that Pasteurella spp. were isolated and phenotypically identified with an

incidence rate of 55% (33 out of 60 rabbits). Ten Pasteurella spp. isolates were randomly chosen for antibiotic

sensitivity testing and molecular identification using PCR. Antibiotic sensitivity test was carried using standard disk

diffusion method against 13 antibacterial drugs to determine antibacterial sensitivity and resistance patterns of

Pasteurella isolates and revealed variable sensitivity and resistance to antibacterial drugs. Pasteurella multocida

isolates were sensitive to wide variety of antibiotics (norfloxacin, enrofloxacin, ciprofloxacin, florfenicol,

doxycycline, gentamycin, cephradine and cefoxitin). Three out of ten isolates were molecularly confirmed to be

Pasteurella multocida and all of them demonstrated the presence of toxA virulence genes. In conclusion, the

prevalence of Pasteurella infections in rabbits in Assiut Governorate was relatively high.

Key words: Antibacterial resístanse Pasteurella multocida, toxA gene, virulence genes.

opportunist or secondary pathogen which can be found in

the respiratory tract of both healthy and diseased animals.

In rabbitries, it could emerge as a major pathogen that

causes upper respiratory tract infections resulting in

considerable economic losses (Deeb and DiGiacomo,

2000 ). Many researchers have distinguished various cases

of non-infected, resistant and chronically infected animals

or even healthy carriers (DiGiacomo, et al., 1983; Deeb, et

al., 1990 ). Pasteurella multocida has been shown to

adhere to the mucosal epithelium of the nasopharynx of

rabbits by fimbriae (pili) which correlated to the virulence

of the organism (Glorioso et al., 1982). A complex

pathogenicity of P. multocida has been reported, and

several virulence factors including hemagglutinins,

fimbriae, lipopolysaccharides, hyaluronidase, iron

regulated protein, capsule, iron acquisition proteins and a

dermonecrotic toxin have been identified previously (Deeb

and DiGiacomo, 2000 ). Previous reports could not detect

toxA gene in P. multocida isolated from rabbits (Ferreira et

al., 2012 ). In addition, other investigators mentioned that

INTRODUCTION

Rabbit pasteurellosis is a serious disease which causes a

considerable economic loss in rabbit production units

(Stelian et al., 2011 ). It is caused by Pasteurella multocida

which may cause pneumonia and septicemia leading to

death or local infections such as rhinitis, otitis media,

conjunctivitis and abscesses (Deeb et al., 1990 ).

Pasteurella multocida is an important pathogen of

the upper respiratory tract of various wild and domestic

animals (Loubinoux et al., 1999). It is a small, Gram-

negative, coccobacillus, non-motile, non-spore forming

and facultative anaerobe which belongs to family

Pasteurellaceae. Pasteurella multocida has been isolated

from all ages of rabbits and colonized most commonly in

the sinus, middle ear, trachea and lungs (Quinn et al.,

1994 ).

Pasteurella multocida is the most common pathogen

isolated from rabbits, its prevalence rate has been recorded

to range from 7-100% (Nakagawa et al., 1986; Deeb, et

al., 1990; Kawamoto et al., 1990 ). It is considered an

To cite this paper: Mohamed FM_,Mansy MF_,Abd-Al-Jwad AM and Hassan AK (2020). Antibacterial Sensitivity and Detection of Virulence Associated Gene of Pasteurella

multocida Isolated from Rabbits. J. World Poult. Res., 10 (2S): 165-171. DOI: https://dx.doi.org/10.36380/jwpr.2020.21

165

Mohamed et al., 2020

toxA gene is not commonly found in P. multocida strains

Phenotypic identification

To confirm the presence of Pasteurella on suspected

samples, Gram’s stain was used for staining films from

bacterial isolates for morphological characters and staining

reaction as a first step for isolates identification and

differentiation. In addition, biochemical reactions;

including catalase, urease and indole tests and

fermentation of sugars tests including dextrose, sucrose,

maltose, dulcitol, arabinose, xylose, lactose, mannitol,

galactose and salicin (Cruickshank et al., 1975 ) were used.

Pasteurella isolates were preserved at 30% glycerol sterile

solution (Biyashev et al., 2014 ).

isolated from rabbits (Ewers et al., 2006; Garcꢀa -Alvarez

et al., 2015; Massacci et al., 2018 ).

Pasteurellosis in rabbits could be controlled through

treatment with antibiotics and the slaughter of infected

animals. Treating infected animals, however, only

alleviates clinical signs and slows the progression of the

disease, but it does not eliminate the infection (Deeb and

DiGiacomo, 2000 ). Vaccination of P. multocida have been

also

reported

using

inactivated

formalized P.

multocida vaccine (Nassar et al., 2013). Methods of

detection and diagnosis of P. multocida infections relied

on microscopic detection of the pathogen via stained

smears, isolation by culturing on selective media, then

phenotypic or characterization serologically (Christensen

and Bisgaard, 2010 ).

Antibacterial sensitivity and resistance patterns

Sensitivity of Pasteurella isolates to antibacterial

agents was determined using standard disk diffusion

method. The criteria proposed by the National Committee

for Clinical Laboratory Standards (CLSI, 2013 ) was used

to determine susceptibility rates. The antibiotic discs

(Oxoid) used in current study were: erythromycin (15µg),

amoxicillin (30 µg), cephradine (30 µg), colistin (10 µg),

ciproﬂoxacin (5 µg), enroﬂoxacin (5 µg), cefoxitin (30

µg), gentamicin (10 µg), neomycin (10 µg), streptomycin

(10µg), florfenicol (15 µg), doxycycline (30 µg), and

norfloxacin (10 µg). Interpretation of results was

performed according to Quinn et al. (1994) and Koneman

et al. (1997).

This study aimed to detect and identify P. multocida

phenotypic

characterization

and

molecular

identification in rabbits as well as to determine its

antibacterial sensitivity and resistance patterns. In

addition, this study investigated the occurrence of toxA, a

gene associated with the virulence, in P. multocida.

MATERIALS AND METHODS

Ethical approval

The research protocol was reviewed and approved by

Institutional Animal Care and use Committee (Vet

CU20022020160).

Multidrug resistant Index

The multi-drug resistance index (MDRI) was

determined for every isolate using the following equation

according to Chandran et al. (2008):

Sample collection

A total of 40 recently dead rabbits with a previous

respiratory manifestation (snuffling) were used to collect

samples from liver, lung and subcutaneous abscesses. In

addition, 20 diseased rabbits were used to collect tracheal

swabs. All rabbits used ranged from 8 to 48 weeks old,

and were obtained from different farms in Assuit

Governorate, Egypt. Samples were collected under

complete aseptic conditions in sterile tubes containing

nutrient broth, transferred immediately to the lab in an

icebox for bacteriological examination.

Number of antibiotics resisted

MDR index =

x 100

Total number of antibiotics used

When an isolate was resistant to more than three

antibiotics, it was considered as multidrug resistant.

Isolates were considered highly resistant when they had

MDRI values of more than 0.2 (20%).

Molecular identification of Pasteurella isolates

Ten randomly selected phenotypically identified

Pasteurella isolates (five from dead rabbits and five from

diseased rabbits) were subjected to molecular

identification using PCR as following:

Isolation of Pasteurella multocida

The collected samples were inoculated into brain

heart infusion broth (BHI) and incubated for 24 hours at

37°C. Sub-culturing was carried out on sheep blood agar

(5%) then incubation for 24 hours at 37°C. Growth was

examined for typical P. multocida colonies.

DNA extraction

DNA was extracted from the samples by using

QIAamp DNA Mini kit (Qiagen, Germany, GmbH)

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J. World Poult. Res., 10(2S): 165-171, 2020

according to manufacturer’s recommendations. In brief,

Pasteurella isolates were obtained from 60 examined

rabbits with overall incidence of 55%. The isolation of

Pasteurella from lung, liver and subcutaneous abscesses

samples from freshly dead rabbits revealed that 25 rabbits

were positive for Pasteurella (62.5%) while the

examination of tracheal swab samples from diseased

rabbits showed an isolation rate of 40% (8 out of 20).

200µl of the sample suspension were incubated with 10µl

of proteinase K and 200µl lysis buffer at 56^OC for 10

minutes. Then after, 200µl of 100% ethanol were added to

the lysate. The sample was then washed and centrifuged.

Nucleic acid was eluted in 100µl of elution buffer that was

provided in the kit.

Oligonucleotide primer

Phenotypic identification of Pasteurella isolates

The growth of suspected P. multocida on brain heart

infusion (BHI) agar, appeared as round gray large mucoid

colonies. On 5% sheep blood agar, colonies were non-

hemolytic dew drop like. Gram stained slides of suspected

colonies showed gram negative rods. The suspected P.

multocida colonies were positive for catalase and indole,

but negative for urease. However, sugar fermentation

revealed that P. multocida colonies were positive with

lactose, sucrose, dextrose, mannitol and galactose, but

negative with dulcitol, salicin, arabinose, maltose and

xylose.

The used primers (table 1 ) were supplied from

Metabion (Germany).

PCR amplification

PCR primers were used in a 25-µl reaction that

contained 12.5µl of EmeraldAmp Max PCR Master Mix

(Takara, Japan), 1µl from each primer of 20 pmol

concentration, 4.5µl of water, and 6µl of DNA template.

The reaction was then carried out in an applied biosystem

2720 thermal cycler.

Analysis of the PCR products

By electrophoresis, the products of PCR were

separated on 1% agarose gel (Applichem, Germany,

GmbH) at room temperature in 1xTBE buffer using

gradients of 5V/cm. 20µl of the products was loaded in

each gel slot for gel analysis. Gelpilot 100bp DNA ladder

(Qiagen, Germany, GmbH) was then used to determine the

sizes of fragments. Photographing of the gel was done on a

gel documentation system (Alpha Innotech, Biometra) and

the data were then analyzed by computer software.

Antibacterial susceptibility and resistance

patterns

Results revealed that all Pasteurella spp. samples

were sensitive to norfloxacin, enrofloxacin, ciprofloxacin,

florfenicol, doxycycline, gentamycin, cephradine and

cefoxitin. The isolates showed MDRI rang from (15.35%

to 42.86%). The Sensitivity and resistance of P. multocida

isolates to erythromycin, amoxicillin, colistin sulfate,

neomycin and streptomycin were variable, results were

summarized in table 2.

Detection of toxA gene

Ten samples of Pasteurella spp. which were

previously identified, were subjected to PCR to detect

toxA gene. DNA extraction from samples was carried out

as previously mentioned. The used primers (Table 1 ) were

supplied by Metabion (Germany). The PCR reaction was

performed according to Townsend et al. (1998).

Molecular identification of Pasteurella multocida

suspected isolates

Total ten samples were randomly chosen for

molecular identification using PCR. Three were confirmed

as P. multocida and seven isolates were not confirmed

(Figure 1 ).

RESULTS

Detection of toxA virulence gene

Expression of toxA virulence gene was demonstrated

in the three isolates that confirmed molecularly as P.

multocida (Figure 2 ).

Isolation rate of Pasteurella multocida

Examination of individual diseased (20) and freshly

dead (40) rabbits showed typical clinical signs and post

mortem lesions of pasteurellosis. The results revealed 33

167

Mohamed et al., 2020

Table 1. Primers sequences, target genes, amplicon sizes and cycling conditions

Amplification (35 cycles)

Amplified

segment

(base pair)

Target

gene

Primers sequences

Primary

denaturation

Final

extension

Reference

Secondary

(5’- 3’)

Annealing

Extension

denaturation

F: ATC-CGC-TAT-TTA-CCC-AGT-

94˚C

5 min.

94˚C

30 sec.

55˚C

40 sec.

72˚C

45 sec.

72˚C

10 min.

Kmt1

460

864

OIE (2012)

R: GCT-GTA-AAC-GAA-CTC-

GCC-AC

94˚C

5 min.

94˚C

30 sec.

55˚C

40 sec.

72˚C

45 sec.

72˚C

10 min

Townsend

et al. (1998)

toxA

F: CTTAGATGAGCGACAAGG

F: forward, R: reverse

Table 2. Antibacterial sensitivity test of Pasteurella multocida isolated from rabbits by disk diffusion method

Results

Isolate

MDRI

FFC

30.07%

15.35%

35.71%

42.86%

30.07%

35.71%

15.35%

35.71%

ER: erythromycin, AX: amoxicilline, CT: colstine sulfate, CP: ciprofloxacin, EN: enrofloxacin, CO: cefoxitin, CN: gentamycine, NO: neomycine, SP:

sterptomycine, FFC: florfenicol, DO: doxycycline, NR: norfloxacin, CE: cephradine, MDRI: multi-drug resistance index. S: sensitive, R: resistance.

Figure 1. Agarose gel electrophoresis showing amplification of 460 bp fragments specific for Pasteurella multocida of 10

samples isolated from rabbits. Lanes 1-6 and 9 are negative, Lane 7, 8 and 10 are positive samples for kmt1 gene. L: 100bp

ladder (100-1000bp). Pos: positive control, Neg: negative control

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J. World Poult. Res., 10(2S): 165-171, 2020

Figure 2. PCR results for 864bp toxA gene of Pasteurella mutocida showing positive amplification of the gene in tested

samples isolated from rabbits. Lanes 7, 8, and 10 are positive samples for toxA gene, Pos: positive control, Neg: negative

control. L: 100bp ladder (100-1000bp).

resistance was common against erythromycin, amoxicillin,

colistin sulfate, neomycin and streptomycin. Unlikely,

Tang et al. (2009) and Ferreira et al. (2012) reported that

the high resistance of the isolates was against

sulfonamides and cotrimoxazole. They reported that

increased resistance of P. multocida against antimicrobials

could be attributed to the irresponsible use, overuse, and

misuse of antibiotics in rabbitaries.

Molecular identification is the basic method for

identifying of P. multocida strains. Due to their great

discriminatory power, DNA-based identification method

has been established as an effective method in

characterization of P. multocida (Blackall and Miflin,

2000 ). Using PCR as method for characterization, the

current results showed that, three out of ten phenotypically

identified Pasteurella isolates were confirmed as P.

multocida (30%). Similar findings have been obtained by

Mazed et al. (2013), where the molecular detection by

real-time PCR of P. multocida revealed its occurrence in

diseased rabbit samples with an incidence of 29.5%.

DISCUSSION

The relatively high prevalence rate of Pasteurella isolation

in the current study suggests that it is probably an

important pathogen causing high mortality in rabbits.

Previous studies revealed variable incidences of P.

multocida isolation (Lee et al., 1990; Takashima et al.,

2001; Stelian et al., 2011 ). Sanchez et al. (2004) isolated

P. multocida from clinically healthy animals with a

prevalence of 20-90%. However, low values of isolation

incidences were also previously reported for P. multocida

from liver, lungs, spleen, heart-blood and nasal swabs of

rabbits with an incidence ranging from 18.75% to 35.00%

(Mazed et al., 2013 ). Lower values of prevalence (3.4% to

9.4%) have been recorded in diseased rabbits by Asran et

al. (2016). This discrepancy in prevalence rates of P.

multocida could be attributed to method of detection

employed or the locality in which the study was done.

Antimicrobial resistance test for 10 randomly

selected Pasteurella isolates reveled that most of them

showed multidrug resistant to more than two antibiotics

with MDRI ranged from 15.35% to 42.86%. Ferreira et al.

(2012) investigated the antimicrobial sensitivity of P.

multocida isolated from rabbits in Brazil and found that

47.8% of the strains were resistant to at least one of the

tested drugs. However, in the current investigation,

Therapy by using antimicrobial is one of the

preliminary control measures to reduce morbidity and

mortality resulting from P. multocida infections in rabbit

and antibiotics are still the first choice to prevent and

control P. multocida infections (Kehrenberg et al., 2001).

However, the improper and misuse of antibiotics promotes

169

Mohamed et al., 2020

the development of drug resistance (Percy et al., 1984;

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