PREVALENCE AND ANTIMICROBIAL RESISTANCE PROFILES OF SALMONELLAE ISOLATED FROM CHICKEN OFFALS AT SLAUGHTER
SLABS IN ZARIA,
Solomon Ocheja OCHENI
A RESEARCH PROPOSAL PRESENTED AT THE SEMINAR OF STAFF AND POST GRADUATE STUDENTS, FACULTY OF VETERINARY MEDICINE, AHMADU BELLO UNIVERSITY, ZARIA.
PROF. J. K. P. KWAGA (CHAIRMAN)
DR. A. M. WAKAWA (MEMBER)
20TH FEBUARY, 2015
Salmonella is a Gram-negative, non-spore forming, rod shaped, facultatively anaerobic bacterium in the family Enterobacteriaceae. However, they can also live under aerobic conditions. Salmonella lives in the environment and intestinal tracts of warm and cold blooded animals i.e. humans and animals (Todar, 2005).
The genus currently contains just two species, Salmonella enterica (including six subspecies) and Salmonella bongori. Most of the Salmonella isolates from cases of human infection belong to Salmonella enterica subspecies enterica. The genus is also further subdivided into approximately 2,500 serovars (or serotypes), characterised on the basis of their somatic (O) and flagellar (H) antigens (Bennasar et al., 2000).
Among the food-borne pathogens, the genus Salmonella is one of the most common causes of food-borne infections worldwide (Baird-Parker, 1990). Salmonella strains include those that cause enteric fever like S. Typhi and S. Paratyphi and those that are agents of food poisoning like Salmonella Enteritidis and S. Typhinerum, but many others have been shown to cause disease, notably S. Infantis, S. Virchow and S. Newport (Bennasar et al., 2000; Winokur, 2000).
Though the primary habitat of Salmonella species is the intestinal tract of animals such as birds, reptiles, farm animals ( Flores-Abuxapqui et al., 2003), nevertheless they can be spread to other parts of the body such as the spleen, liver, bile, mesenteric and portal lymph nodes (Ezeugoigwe et al., 2014). The worldwide annual estimation of the incidence of non-typhoidal salmonellosis (NTS) is 1.3 billion and annual death is estimated to be 3 million cases (Tassios, 1997).
Poultry and poultry products are imperative elements within the human food chain, but are widely accepted as an important reservoir of intestinal and food-borne pathogens like Salmonella and are recognized as vital sources of Salmonella infection in human (Hughes et al., 2007; Limawongpranee, 1999; EFSA, 2004.). Salmonellosis is considered to be one of the major bacterial disease problems in the poultry industry, world-wide. Salmonella species are responsible for a variety of acute and chronic diseases in both poultry and humans (Majowicz et al., 2010; Okwori et al., 2013) and infected poultry products are among the most important sources for food-borne outbreaks in humans. Isolation of Salmonella is reported more often from poultry and poultry products than from any other animal species (Yan et al., 2003). It has also been isolated from sheep and goats (Falade, 1978; Tabukun, 1990), cattle and dogs (kwaga et al., 1985; Kwaga et al., 1989)
S. Enteritidis and S. Typhinerum are among the most pathogenic strains of Salmonella serovars frequently isolated from poultry and poultry products (Keery, 2010), and a good number of them can be harboured by poultry without showing any clinical signs (Gast, 2007).
Salmonella infections (salmonellosis) in humans often result from the ingestion of contaminated or undercooked foods, such as poultry, beef, pork, eggs, milk, seafood, and fresh produce (CDC, 2006 ; Dalton et al.,1995). Direct contact with animals also results in transmission of Salmonella to humans (Braden, 2006). Though Salmonella mostly contaminate meat surfaces from faecal material during slaughter and processing, sometimes, it may be present internally in meat tissue of infected animals (Braden, 2006). Contamination of poultry meat with Salmonella in slaughterhouses occurs through faeces of symptomatic or asymptomatic animals, contaminated equipment, floor and personnel (Thorns, 2000). The pathogens can survive in the poultry meat until presented to the market (Redmond and Griffith, 2006).
The outbreak of salmonellosis is common in the developing nations especially in Africa, Asia, South and Central America. Analysis carried out showed that there is high prevalence of Salmonella species in birds in Nigeria (Okwori et al., 2007; Ajayi and Egbebi, 2011). A study conducted in 2001 to determine the prevalence of S. enterica serovar Enteritidis in Maiduguri, Nigeria found Salmonella in 27% (n=150) chicken meat samples, of which eight were Salmonella Enteritidis (Ameh et al., 2001).
The routine practice of antibiotic utilization in domestic animals as a means of preventing and treating diseases, as well as promoting growth, is an important factor in the re-emergence of antibiotic-resistant bacteria that are consequently transferred to humans in the course of the food chain (Tollefson et al., 1997; Witte, 1998). In recent times, a significant increase in the occurrence of antimicrobial drug resistance in Salmonella strains is of great concern in both developed and developing countries (Alambedji et al., 2003; Threlfall et al., 2002; Cailhol, 2006).
1.1 STATEMENT OF RESEARCH PROBLEM
Food-borne microbial illnesses are an important public health issue worldwide. It not only affect people's health and well-being, but also have economic impacts on individuals and the countries. It reduces markedly social and economic productivity of countries (Carbas et al., 2012). Although these illnesses are usually a mild to moderate self-limiting gastroenteritis, invasive diseases and complications may occur (Glyn et al., 1998). Many food-borne bacteria colonize the gastrointestinal tracts of a wide range of wild and domestic animals, especially animals raised for human consumption (Banasser et al., 2000). Food contamination with these pathogens can occur at multiple steps along the food chain, including production, processing, distribution, and preparation (Richardson and Mead, 1999; Gillespie, 2002). Salmonella enterica is a common cause of human gastroenteritis and bacteremia, and a wide variety of animals, particularly food animals, have been identified as reservoirs for non-Typhi Salmonella (Doyle, 1987; Humphrey, 2000; Mead, 1999; Adak et al., 2002). However, poultry are often considered the most common reservoir for human Salmonella infection in humans (Braden, 2006). It has been estimated that over 1.4 million cases of salmonellosis occur each year in the United States alone (Galanis et al., 2006). Two populations that are most at risk of food-borne illness are children and immune compromised individuals. The infective dose can be quite low (10-100 cells) in this vulnerable individuals or when contaminated food with a high fat content, like chocolate or cheese, is consumed (Benasser et al., 2000).
An additional concern is the growing incidence of antimicrobial-resistant food-borne pathogens. Food contamination with antibiotic-resistant bacteria can be a major threat to public health (Boonmar et al., 1986). Antimicrobial resistant bacteria from animals, both commensal and pathogenic variants, can reach the general public via exposure to contaminated food products of animal origin if they are improperly cooked or otherwise mishandled. It is known that these resistant bacteria have the potential to colonize humans and/or transfer their resistance determinants to resident constituents of the human microflora, including pathogens (Bensink et al., 1981). Multidrug resistant Salmonella are frequently isolated from food sources in South East Asia. It is hard to treat the infections caused by multidrug resistant bacteria as compared to susceptible. Such strains are more dangerous and of great food safety concern (Adis et al., 2011). The increase in the prevalence of multi-drug resistant Salmonella, particularly resistance to fluoroquinolones and third-generation cephalosporins are an emerging problem worldwide (Hur et al., 2012). The presence of resistant Salmonella in retail poultry meat has been assessed in some studies. In a pilot survey by White et al. (2001) using two hundred ground meat samples (51 chicken, 50 beef, 50 turkey, and 49 pork), Salmonella was isolated more frequently from poultry (33% of chicken and 24% of turkey samples) than red meats (18% of pork and 6% of beef samples). In that study all the isolates were resistant to one or more of the antibiotics tested.
Studies also reported the important role of S. Typhi and NTS in septicaemia in humans in Ibadan, Nigeria. However, the specific NTS serotypes (Alausa et al., 1997) were not revealed, probably due to the lack of resources and high-quality antisera that are often lacking in many African countries (Hendriksen et al., 2009).
A recent study reported high rates of resistance to ciprofloxacin in S. Hiduddify in chickens in Maiduguri, Nigeria (Ameh et al., 2009). Otherwise, there are limited data on the antimicrobial resistance of Salmonella from both humans and food animals in Nigeria.
Poultry is an important food source to man; chicken and turkey meat dishes are special delicacies, particularly during festive periods in Nigeria, but the poultry industry is largely unregulated. Chicken meats comprise about two-thirds of the total poultry meat production in the world (Ruban et al., 2010). Poultry meat is easy to prepare at home and widely used in restaurants and fast-food establishments. There is no primary religious restriction on the consumption of poultry meat (Richardson and Mead, 1999). In the majority of food-borne infections, it is not possible to identify the food vehicle. Poultry meat is considered as the most commonly reported vehicle of food-borne pathogens, followed by the red meat (Hughes et al., 1998). The most common chain of events leading to this food-borne illness involves healthy carrier animals which subsequently transfer the pathogen to humans during production, handling and/or consumption (Stepfoth, 2006). The pathogens are mainly disseminated by trade in poultry and uncooked poultry food products (Gillespie et al., 2005).
Monitoring the presence of pathogens in food is the primary tool for the implementation of food safety systems. It is necessary to monitor the prevalence and antimicrobial resistance of foodborne pathogens for effective food safety planning and targeted interventions (Enabulele et al., 2008; Calleja et al., 1993).
The development and the accumulation of resistance to antimicrobials in food-borne pathogens are a major problem for public health. Multi-drug resistant Salmonella may acquire their resistance genes from microbiota of production animals, especially poultry before being transmitted to humans through food chain (Okoli et al., 2006; White et al., 2001; Threlfall, 2002).
In developed countries, stringent control of antibiotic use coupled with effective surveillance of antibiotic resistance patterns in the population, have successfully reduced the prevalence of antibiotic resistant bacteria (Collignon, 2003). The situation in the developing countries like Nigeria is however different, where antimicrobial agents are readily available to people in local drug stores without prescription (Kwaga and Adesiyu, 1984). Such practice has led to misuse of antibiotics with the associated high prevalence of antibiotic resistance among isolates from animal and food sources (Enabulele et al, 2008).
Due to lack of coordinated epidemiological surveillance programs in most of Africa (Kagambega et al., 2013) including Nigeria, information on the prevalence of Salmonella in chicken meat is limited especially in the northern parts of the country. In the light of the foregoing, this study is aimed at determining the occurrence and antimicrobial resistance of Salmonella isolates in chicken offals (liver, intestine) from slaughter slabs in Zaria.
1.3 RESEARCH QUESTIONS
Are Salmonella found in offals of chickens obtained from slaughter slabs in Zaria and its environs?
If they are found, what are the antimicrobial resistant profiles of the Salmonella isolates?
1.3.1 AIM OF STUDY
The aim of the study is to determine the prevalence and antimicrobial resistance profiles of Salmonellae isolated from chicken offals (liver and intestine) from slaughter slabs in Zaria and its environs.
1.3.2 OBJECTIVE OF STUDY
To determine the prevalence (by isolation, identification and characterization) of Salmonella organisms in offals of chickens slaughtered at slaughter slabs in Zaria and environs.
To determine the antimicrobial resistance profiles of Salmonella isolates if found in chicken offals.
To determine the M.I.C (minimal inhibitory concentration) of ciprofloxacin-CIP and gentamycin-CN.
To detect the presence of InvA gene by PCR method.
2.0 MATERIALS AND METHODS
2.1 STUDY AREA AND SAMPLING SITES
The study will be conducted in Zaria, which is located in the center of Northern Nigeria, on a plateau at 2200 feet above sea level (Mortimore, 1970). It is positioned between Latitude 11˚3'N and 7˚42'E. Its climate is tropical continental characterized by cool, humid wet seasons and cold or hot dry seasons (Mortimor, 1970). The sampling areas will be the slaughter slabs in live bird markets (Samaru market, SabonGari, Tudun Wada, Kwangilla and Dan magaji) which are all located within Zaria. These areas are selected based on convenience and on the fact that large number of birds are slaughtered on a daily bases in these locations.
2.2 SAMPLE SIZE: The sample size to be used for the prevalence study and isolation of Salmonella in this survey will be determined and the sampling will be done on a weekly basis in order to obtain the required and varied number of samples. The sample size to be used is derived using the formula described by Thrusfield (2008).
N = Z2Pq
Where N = sample, Z =1.96, d2 = allowable error (5%), P =anticipated prevalence for Salmonella in poultry offals (24.8%) (Ameh et al., 2009); N=286. This sample size will be the minimum number of offals collected from the slaughter slabs. For the purpose of convenience, the total sample size will be rounded up to 300 samples with 60 samples will be collected from each of the 5 slaughter slabs selected.
2.3 SAMPLE COLLECTION AND PROCESSING
Chicken offals (liver and intestine) samples (n = 300) will be obtained from the various slaughter slabs at live bird markets within Zaria. Samples will be collected from the various breeds of chicken (local, broilers and layers) based on their availability at the different slaughter slabs. Each sample will be collected aseptically and transported in portable coolers containing ice pack immediately to the Bacteria Zoonosis Laboratory of Faculty of Veterinary Medicine, ABU for analysis.
2.4 LABORATORY ANALYSIS
2.4.1 Procedure for examination of presence of Salmonella in samples
From each sample pool, 25 g will be weighed and cut into tiny pieces using sterile scalpel blades and scissors. Isolation and identification of Salmonella will be carried out following the techniques recommended by the International Organization for Standardization (IOS, 1999). Briefly, each 25g sample will be put into a stomacher bag containing 225 ml buffered peptone water (BPW, Merck) and homogenized. The homogenate will be incubated at 37°C for 18 hours. From the pre-enriched sample, 0.1 ml aliquot will be transferred into 10 ml of Rappaport Vassiliadis (RV, Merck) and incubated at 37°C for 24 hours.
2.4.2 Plating: A loopfool of the enrichment broth will be streaked on SSA (Salmonella shigella agar), xylose lysine deoxycholate (XLD) agar and xylose lysine targitol (XLT) agar plates to ensure isolated colonies which will then be incubated at 37°C for 24hours.
2.4.3 Preliminary identification (selective media): One or more characteristic colonies appearing colourless, with or without black centers on SSA agar will be picked and inoculated into Triple sugar Iron (TSI) agar and Urea agar. Colonies which give reactions suggestive of Salmonella by showing alkaline/acid with or without gas and hydrogen sulphide on TSI and are urease negative will be stored at 400C on Nutrient agar (NA) slants until characterized (IOS, 1999).
2.5 IDENTIFICATION AND CHARACTERIZATION OF ISOLATES
2.5.1 Biochemical Identification and characterization of isolates
Characterization will be done based on the standard techniques (Barrown and Feltham, 1995). All isolates that give reactions typical of Salmonella in all or most of the test substrates will be considered to belong to the genus Salmonella. Typical Salmonella reactions are indole negative, methyl red positive, Voges Proskauer negative, citrate positive, motile in motility medium, produces H2S, nitrate positive, lysine decarboxylase positive, oxidase negative, ferments glucose, mannitol and raffinose but fails to ferment sucrose and lactose.
2.5.2 Microbact 12F Gram-nagative bacillus (GNB) Rapid identification System
A 24-hour culture of presumptive Salmonella colonies on selective media will be obtained after which and oxidative test will be performed using oxidase test strips. One to three (1-3) isolated colonies of the isolates will then be selected and emulsified in 3ml sterile normal saline. The plate will be placed in a holding tray and the seal pulled back. Four drops of bacterial suspension will be added to each well, resealed and incubated at 37°C for 24hours, after which appropriate reagents are added to wells 8, 10 and 12. Two drops of Kovac's reagent will be added to well 8 and observed for 2 minutes, 1 drop each of Vp1 (Voges Proskauer) and Vp2 to well 10 and observed for 10-30 minutes and 1drop of TDA reagent in well 12, which will be interpreted immediately.
Results will be recorded in report forms containing the substrates that are tested. Twelve substrates will be tested; lysine, ornithine, hydrogen sulphide, glucose, mannitol, xylose, ONPG (O-Nitrophenyl 1 B-D galactopyranoside), indole, urease, Voges Proskauer, citrate and TDA. Three substrates will form one group with each substrate assigned a number. When a substrate gives a positive result, the corresponding number for that group is summed up and recorded. The result of the corresponding 3 wells will be added and a 4 digit code will be obtained and fed into the computer identification software. This will give the probable identity of the organism tested as percentage probability. The microbact software permits a 75% cut-off point for a probable identification. All test organisms that give less than 75% will not be accepted as Salmonella.
2.6 CONFIRMATORY TEST FOR SUSPECTED SALMONELLA ISOLATES
Isolates suspected to be Salmonella will be serologically tested using Salmonella polyvalent 'O' group A-Z antiserum latex kit according to the instruction of the manufacturer (OXOID). All isolates will be streaked on Salmonella-Shigella agar plates and incubated at 37°C for 24hrs. Latex reagents will then be brought to room temperature. One drop of the latex reagents will be dispensed onto a circle on the reaction card, and a drop of saline will placed into the circle distant from the latex. Using a loop, a portion of the colony of presumptive Salmonella species on Salmonella-Shigella agar plates will be emulsified in the saline drop on a portion of the circle on the card. The test latex and the resulting smooth suspension will be mixed together and spread to cover the reaction card using a loop. The card will be rocked in a circular motion observing for agglutination within 2minutes.
2.7 EVALUATION OF THE IN VITRO SUSCEPTIBILITY OF THE ISOLATES TO ANTIMICROBIAL AGENTS
All the isolates will be tested for their susceptibility to 12 antimicrobial agents with the following disc contents; tetracycline TE (30ug), streptomycin S (10ug), amoxicillin / clavulanic acid-AMC (30ug), kanamycin K (30ug), chloramphenicol C (30ug), penicillin G P (10IU), trimethoprim W (5ug), sulphamethoxazole/thrimethoprim SXT (25ug), gentamicin- CN (10ug), ciprofloxacin-CIP (5ug), nitrofurantoin F (50ug) and neomycin N (10ug), by the disk diffusion method (CLSI, 2009).
Two to three (2-3) colonies of the appropriate culture will be inoculated into 5ml tryptone soy broth and incubated at 370C until the turbidity approximates 0.5 McFarland's standards.
Mueller–Hinton agar plate will then be prepared and used according to manufacturer's instruction. Sterile swabs will be dipped into the broth culture with the excess broth drained by pressing on the inner side of the tube and used to streak the Mueller–Hilton agar in 3 directions at 180°C until the entire surface is streaked. Control strains will also be incorporated as part of organisms tested. The plates will be allowed to dry at room temperature for 10minutes and the antimicrobial disc dispensed into the plates using disc dispenser (Oxoid) and further pressed with forceps to ensure complete contact with medium.
The Petri dishes will be inverted and incubated at 37°C for 18hours. Following incubation, the zones of inhibition will be measured to the nearest millimeter and interpreted based on interpretation of zone diameter of test culture provided by Clinical and Laboratory Standards Institutes (2011).
2.8 M.I.C DETERMINATION (CIPROFLOXACIN-CIP AND GENTAMYCIN-CN)
The MIC-determination method will be in accordance with international recommendations by the Clinical and Laboratory Standards Institute (CLSI, 2009). Sensitivity of the isolates to the antimicrobial agents is done by agar dilution method in Mueller-Hinton agar media to determine the minimum inhibitory concentrations (MICs). The antimicrobial agents (CIP and CN) M. I. C evaluator strips used will be obtained from Oxoids.
2.9 DETECTION OF InvA GENE BY PCR METHOD
2.9.1 DNA extraction of isolated Salmonella:
Bacteria will be cultured on LB (Luria Bertani) agar for 24 hrs at 370C. DNA extraction will be carried out directly from the isolates by boiling 1.5 ml aliquots of isolates in broth for 5 minutes and the supernatant following sedimentation at 12,000g for 5 minutes will be used as source of DNA. The concentration of extracted DNA will be estimated using a Nanodrop spectrophotometer (Nanodrop; Pretoria, South Africa).
2.9.2 Primer set and PCR amplification program:
Salmonella specific primers, Salm3 and Salm4 (Cocolin et al, 1998) have the following nucleotide sequence based on the targeted invA gene of Salmonella; 5´ - GTG AAA TTA TCG CCA CGT TCG GGC AA - 3´and 5´ - TCA TCG CAC CGT CAA AGG AAC C -3' for forward and reverse primers respectively. PCR will be carried out using the above primers in a 25 μl reaction mixture containing 1 x PCR buffer (Promega, Madison, WI), 1.5 mM magnesium chloride, 200 μM of each dNTP, 20 pmol of each primer, 1.25 U Taq DNA polymerase (Promega, Madison, WI). The DNA will be diluted to give a final concentration of between 10-200 ng/μl and 1μl will be used as a template in the PCR. Amplification to be carried out in an Eppendorf Mastercycler Gradient (Hamburg, Germany) using the following cycling condition; initial denaturation at 95ºC for 5 minutes, 35 cycles of 95ºC for 1 minute, 65ºC for 1 minute and 72ºC for 90 minutes and a final extension step of 72ºC for 10 minutes.
PCR amplicons are to be separated by 1.5% agarose gel electrophoresis at 80volts (stained with ethidium bromide and visualized by UV illumination) and photographed. 50bp DNA size markers will be used for calibration. This will serve as the expected amplicon size.
2.9 DATA ANALYSIS
The prevalence of Salmonella will be calculated by dividing the total number of samples positive, by the total number of samples collected, and then expressed as a percentage. Results will be presented using tables and graphs. Data obtained during the study will be analyzed using Statistical Package for Social Science (SPSS version 17.0; SPSS inc. Chicargo, IL, USA). Chi-square test or Fisher's exact test, where necessary will be performed in order to show the relationship between the variables like: Salmonella occurrence (%), % susceptibility or resistance of Salmonella to antimicrobial agent, % prevalence of the different Salmonella strains, detected and characterized in poultry. Values of P<0.05 will be considered significant.
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