DETECTION OF SOME VIRULENCE GENES IN SALMONELLA SPECIES ISOLATED FROM DUCKS AND DUCK EGGS

Assiut University web-site: www.aun.edu.eg

DETECTION OF SOME VIRULENCE GENES IN SALMONELLA SPECIES ISOLATED FROM DUCKS AND DUCK EGGS

EL-GAOS, M.I.; KHALIL, M.R. AND GHADA ALLAM ABD EL-DAYEM

Poultry Diseases Dept., Animal Health Research Institute, Mansoura

Received: 29 October 2019;     Accepted: 26 November 2019

INTRODUCTION

Salmonellosis is a zoonotic bacterial disease of national and international importance. The worldwide distribution of Salmonellosis often parallels the patterns of trade of animal products and food, and the migration patterns of human and animals (Gilbert      et al., 2010). 

Avian Salmonellosis is an important disease causing serious impediment to the development of poultry industry especially in developing countries of Asia and Africa. Since no "effective" immuno-prophylactic measures are available for the disease till now, strict biosecurity is the only alternative to preclude the disease (Rajagopal et al., 2013).

Corresponding author: EL-GAOS, M.I.

E-mail address: elgaos122@gmail.com  

Present address: Poultry Diseases Dept., Animal Health Research Institute, Mansoura

The pathogenicity of Salmonella depends on a series of factors associated with the bacterium, the bird itself and the conditions in which the bird is raised. Attachment and penetration of the bacterium into digestive mucosa is a prerequisite for systemic infection (Rychlik et al., 1999). Polymerase chain reaction (PCR) is molecular biology technique which has taken up an increasingly significant space in the field of laboratory diagnostics, allowing the detection of various pathogens such as Salmonella species in different kinds of food. PCR can reduce the time required to detect and identify the agent with high specificity and sensitivity (Santos et al., 2001).

Most bacterial pathogens including pathogenic Salmonella species have multiple virulence properties that enable them to invade and survive within the host and ultimately cause a disease (Marcus et al., 2000).

Detection and monitoring of drug-resistant Salmonella are important to substantiate the choice of antibiotics for the treatment of clinical Salmonellosis and to assess the risk of exposure of multiple drug resistant strains (Yang et al., 2002).

This study was planned to estimate the prevalence of salmonella serovars implicated in the majority of infection in ducks and duck eggs and todeterminesome virulence genes associated with S. typhimuriuminfection in addition to study antimicrobial resistance profile that help to reduce the spread of resistant salmonella among the diseased ducks via planning a proper control program.

MATERIALS AND METHODS

 Samples:

A total number of (450) samples from ducklings, ducks and duck eggs were collected as shown: (200)ducklings samples from internal organs (liver, caecum, gall bladder and spleen) of (50) ducklings, 1-3 weeks old (freshly dead, diseased living and apparently healthy birds), (200) ducks samples from internal organs (liver, caecum, gall bladder and spleen) of (50) ducks (freshly dead, diseased living and apparently healthy birds) and (50) unhatched duck eggs (dead in shell embryo). All samples were obtained from different duck farms and hatcheries located in Dakahlia Governorate under aseptic condition in ice box and transferred to the laboratory. Collected samples were cultured within a limit time.

Clinical and Postmortem examination:

All birds were examined clinically, then sacrificed and immersed in disinfectant before being autopsied. Gross pathological changes were recorded, summarized and presented with results for both freshly dead and clinically sick ducks.

Isolation of salmonellae:

The collected samples were transferred to test tubes containing Tryptose broth and Selenite F-broth and incubate at 37 ^oC for 18-24 hours, followed by subculturing on blood agar, MacConkey’s agar and Xylose lysine desoxycholate agar plates and incubated aerobically at 37 ^oC for 18-24 hours ISO 6579 (2002).

Biochemical identification of the isolates:

The growing colonies on various plates were examined morphologically, culturally and biochemically (Indole, Nitrate Reduction, Voges Proskaur, Citrate Utilization, Urease, Sugar Fermentation and Coagulase test) Quinn et al. (2002).

Serological identification of salmonellae:

Serological identification of purified strains ofSalmonella using available agglutinating Salmonella test sera (Denken Selken Co. LTD, Tokyo, Japan) according to Kauffmann (1973).

PCR technique for detection of virulence genes (Oliveira et al., 2003):

PCR test was carried out for seven S.typhimurium samples in PCR unit in Animal Health Research Institute, AHRIfor detection of virulence genes. PCR amplification kit the QIAamp®DNA Mini Kit (Cat. No. 51304-Qiagen) was used according to manufacturer’s instructions.

DNA extraction:

Chromosomal DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56 ^oC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100µl of elution buffer provided in the kit.

Oligonucleotide Primer:

Primers used were supplied from Metabion (Germany) are listed in Table (1).

PCR amplification:

Primers were utilized in a 25- µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in a T3 Biometra thermal cycler.

Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the products was loaded in each gel slot. A gelpilot 100 bp DNA Ladder (Qiagen, Germany, GmbH) and generuler 100 bp ladder (Fermentas, Thermo) were used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

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

In vitro antibiotic susceptibility test:

Seventeen S.typhimurium isolates were subjected to antibiotic sensitivity test against 15 commonly used antibiotics. The antimicrobial susceptibility profile against oxytetracycline, ampicillin, erythromycin, gentamycin, streptomycin, neomycin, lincomycin, chloramphenicol, ciprofloxacin, trimethoprim-sulfamethoxazole, amoxicillin, doxycycline, flumequine, colistin and norfloxacin were tested by disk diffusion methods according to Clinical and Laboratory Standards Institute (CLSI, 2012).     

RESULTS

Clinical and postmortem findings of examined birds:

The clinical signs observed on examined ducks suffered from salmonellosis were lowered heads, closed eyes, drooping wings, ruffled feathers, Whitish watery diarrhea, death, pasted vent, conjunctivitis, retarded growth and lameness.

The postmortem examination of both freshly dead and sacrificed ducks suffered from salmonellosis wereunabsorbed yolk sac (ducklings), congested internal organs (Liver, kidney and spleen) with enlarged gall bladder, pinpoint necrotic foci on the bronzed liver, spleenomegaly with congested kidneys and white cecal core with enteritis.

Table 2: The prevalence of Salmonella species recovered from collected samples.  

Table 3: Serotyping of the isolated Salmonella species.

S. typhimuriumwas the most prevalence isolates

Table 4: Antibiotic sensitivity and resistance pattern for (17) S.typhimurium isolates.  

Table 5: Result of PCR assay for detection of virulence genes of S. typhimurium.

Figure (1): Agarose gel electrophoresis showing amplification of 467bp fragment using bcfc primer and 517 bp fragment using sopB primer.

L: 100- 600 bp ladder.

Lane (1-7): positive samples.

Neg: negative

Pos: positive

Figure (2): Agarose gel electrophoresis showing amplification of 677bp fragment using mgtC primer and 284 bp fragment using invA primer.

L: 100- 1000 bp ladder.

Lane (1-7): positive samples.

Neg: negative                               

Pos: positive

DISCUSSION

Salmonella is one of the most important pathogen that can infect poultry and causing serious illness in human through consumption of contaminated food products. Egypt has experienced the repeated introduction and dissemination of a wide range of Salmonella serotypes that have become a major concern for both human and animal health. Detection of Salmonellosis is considered a good tool in protection of poultry industry FAO (1994). Further more, Salmonella infection is one of the most important bacterial diseases in poultry causing heavy economic loss through mortality and reduced production Haider et al. (2004).

The present investigation revealed that the clinical signs of salmonellosis in ducks were lowered heads, closed eyes, drooping wings, ruffled feathers, whitish watery diarrhea, death, pasted vent, lameness and conjunctivitis. These findings are in agreement with those observed byRiberio et al. (2005).

Postmortem lesions of salmonellosis recorded in ducks were unabsorbed yolk sac, congested internal organs, pinpoint necrotic foci on the liver with bronze discoloration, spleenomegaly with congested kidneys and white cecal core. These findings are similar with those observed by Saif et al. (2008).

In our study 71 out of 450 samples from ducklings, ducks and duck eggs were found to be positive to Salmonella (15.8 %) as shown in table (2). This result was in agreement with that of Lam et al. (2002) and Nayera(2012) who reported (12.7%) and (14.42%), respectivily. Meanwhile, Asawyand Abd El-latif(2010) reported (3.3%) and Abd El-Tawab et al. (2015) reported (9.6%). Twenty nine samples from ducks were found to be positive with a percentage of (14.5 %). This finding was in accordance with that of Mondai et al. (2008) who recorded (13.07%). On the other hand El-Zeedy et al. (2007) and Tasiand Hsing (2005) recorded (7.8%) and (4.6%) for Salmonella, respectivily.

In the current study 37 ducklings samples were found positive for Salmonella isolation at a percentage of (18.5 %). This result was in the same direction with Huang et al. (1994) who reported (19.3%) and Lam et al. (2002) (18%). This result disagreed with Osman et al. (2014) who reported (12%). The prevalecce of isolation of Salmonella in ducklings (18.5%) was higher than that in ducks (14.5%). This was in accordance with that of Lam et al. (2002) and Tasi and Hsing (2005).

In we present study five duck eggs were positive for Salmonella isolation at rate of (10 %). This result agreed more or less with the results reported by Shareef et al. (1997) and Mohammed(2014) who reported a prevalence rate of (8%) and (9.09%), respectively. In the current study the isolated Salmonella serotypes from ducklings were S.kentuckey, S.infantis, S.typhimurium, S. newport and S.enteritidis as shown in Table (3) These findings were in agreement with that reported by El-Zeedy et al. (2007) who isolated S.enteritidis, S.typhimurium and S.infantis from ducklings. Meanwhile the isolated Salmonella serotypes from ducks were S.derby, S.newport S.typhimurium, S.kentucky, S.enteritidis and S.shubra. Similar results for serological identification were reported byTasi and Hsing(2005) who isolated S.typhimurium, S.newport and S.derby from ducks. The isolated Salmonella from duck eggs were S.typhimurium, S.infants, S.enteritidis and S. newport. Mohammed (2014) was able to isolated S.typhimurium from duck eggs.

 In the present study S. typhimurium was the most prevalence isolates. This might run parallel with Mituniewicz et al. (2007). But Hamad(2017) reported that S.enteritidis was the most prevalent isolates from ducks samples.

Regarding antibiotic sensitivity our study showed that S.typhimurium was highly resistant to streptomycin, lincomycin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole in percentage of 100%, 88%, 82%, 76% and 70%, respectively as shown in table (4). This result nearly agreed with that recorded by Yoshida et al. (1993) who cited that Salmonella was resistant to oxytetracycline, streptomycin and trimethoprim-sulfamethoxazole and Oja andAdetosoye(2009) who reported that, S.typhimurium was resistant to   erythromycin. In the present study S.typhimurium showed moderate resistance to neomycin (59%), gentamycin (59%), amoxicillin (53%), ampicillin (53%) and chloramphenicol (47%). This is in agreement with Mondai et al. (2008) and Boris et al. (2012). The highest sensetivity rates were (59%, 65%, 82%, 94% and 94%) for doxycycline, flumequine, norfloxacin, ciprofloxacin and colistin, respectively. This was in the same direction with Chashni et al. (2009)who reported that the sensitivity rates of S.typhimurium were to flumequine (74%) and norfloxacin (93%),Abd El-Hamid et al. (2003) norfloxacin (86.96%) and Nagappa et al. (2007) showed that colistin was highly effective aganist S.typhimurium. On the other hand Cardoso et al.(2006) reported that S.typhimurium was resistant to colistin.

Amplification of invA gene now has been recognized as an international standard for detection of Salmonella genus Malorny et al. (2004). The invA gene encodes a protein in the inner membrane of bacteria, which is necessary for invasion to the epithelial cells of the host Darwin and Miller(1999). Development of a PCR system remains a suitable molecular tool to diagnose Salmonella on the basis of invAamplification and this has been reported by Bisi-Johnson et al. )2011).

In the present study, PCR assay was carried out for the detection of the invA gene from the isolated strains. The result revealed that the gene was present in all of the isolates (100%) that was demonstrated by the presence of 284 bp PCR amplified fragment which agreed with Dione et al. (2011).

Concerning bcfC gene (fimberial gene) code for an outer membrane usher protein, which carried in the microbial genome as part of a transposable element allowing a better bacterial protein secretion during the intestine invasion and present in all strains with (100%).This result agreed with that of Nayera (2012) who present it in almost strains (95.7%). Regarding sopBgene encodes an inositol phosphate phosphatase that is transported into epithelial cells by the SPI-1 secretion system and indirectly affects chloride secretion by inositol phosphate signaling pathways. PCR assay was carried out for the detection of the sopBgene from the isolated strains. The result revealed that the gene was present in all of the isolates (100%). This was in accordance with that of Mohammed(2014). With respect to mgtC gene is required for intra-macrophage survival and growth in magnesium-depleted medium and also is essential for organisms that invade host cells as cited by Alix and Blanc-Potard(2008),  the mgtC gene was present in all examined isolates(100%). This result was similar to that of Zou et al. (2012). In the same regard Nayera (2012) and Mohammed (2014) were able to find mgtC gene in (54.3%) and (53.3%) of examined strains,respectivily.

The current investigation concluded that, there was significant association between invA, mgtC, sopB and bcfC virulence genes and pathogenicity of S.typhimurium which expresed high multiple antibiotic resistance which require strict regulations of using antibiotics in duck farms to minimize the resistant bacterial stranis.

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