© Springer Science+Business Media New York 2015
Mónica V. Cunha and João Inácio (eds.)Veterinary Infection Biology: Molecular Diagnostics and High-Throughput StrategiesMethods in Molecular BiologyMethods and Protocols124710.1007/978-1-4939-2004-4_2222. Characterization of Campylobacter jejuni and Campylobacter coli Genotypes in Poultry Flocks by Restriction Fragment Length Polymorphism (RFLP) Analysis
(1)
iMed.UL—Research Institute for Medicines and Pharmaceutical Sciences, Intracellular Trafficking Modulation for Advanced Drug Delivery, Faculdade de Farmácia, Universidade de Lisboa, Avenida Prof. Gama Pinto, 1649-003 Lisboa, Portugal
(2)
Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Rua General Morais Sarmento, 1500-311 Lisboa, Portugal
(3)
Centro de Biologia Ambiental, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Lisboa, Portugal
Abstract
We describe a simple, rapid, and discriminatory methodology that allows the routine molecular characterization of Campylobacter jejuni and Campylobacter coli isolates. The proposed approach is built on one of the earliest and simplest molecular typing methods ever, consisting on the analysis of the fragments of different lengths generated by digestion of homologous DNA sequences with specific restriction endonucleases, a process known as restriction fragment length polymorphism (RFLP) analysis. The strategy underneath the workflow reported here is meant to explore the polymorphisms of Campylobacter spp. flaA gene (flaA-RFLP) that allows the local investigation of the genetic diversity and distribution of C. coli and C. jejuni isolates from different sources, namely, chickens’ caeca. Although not appropriate for global and long-term epidemiological studies as a single approach, flaA-RFLP analysis can be very useful in surveys limited in space and time and, for specific epidemiological settings, an alternative to more modern and resource-demanding techniques.
Key words
GenotypingRestriction fragment length polymorphism flaA-RFLP Campylobacter jejuni Campylobacter coli Molecular epidemiology1 Introduction
Campylobacteriosis caused by Campylobacter spp. is considered one of the most prevalent zoonotic enteric infections occurring worldwide [1], Campylobacter jejuni being associated to the vast majority of human campylobacteriosis cases (about 90 %), followed by C. coli (5–10 %) [2, 3]. The main reservoirs of these species are the gastrointestinal tracts of poultry (e.g., chickens, turkeys, ducks, and geese). Handling and consumption of chicken concur as important risk factors in pathogen transmission to humans [4–7].
Isolation, identification, and phenotypic and genetic characterization of pathogens are fundamental to understand the epidemiology of infectious diseases. Using suitable genotyping methodologies, essential information on the sources and routes of pathogen infection may be gathered, which may help to prevent and control infectious diseases and to minimize the occurrence and/or effects of potential outbreaks.
Several methodologies have been developed to genotype Campylobacter spp. [8, 9]. However, the procedures applied in different laboratories, both at the national and international levels, lack in standardization. Monitoring prevalence and distribution of different strains at a local, national, and global level is, therefore, hampered [10]. Efforts have been made in this matter, particularly in the United States, through the implementation in 1996 of the PulseNet network (http://www.cdc.gov/pulsenet/). This network is responsible for the standardization of methodologies applied in the subtyping of food-borne pathogens. In the European Union, the Campynet (http://campynet.vetinst.dk/CONTENTS.HTM) network was established in 1998 aiming the uniformization of the methods used in C. coli and C. jejuni subtyping. Despite these efforts, the progress made in the standardization of Campylobacter spp. subtyping has been occurring slowly [10].
In this report, we describe a simple procedure based on the restriction fragment length polymorphism analysis (RFLP) of the flaA gene (flaA-RFLP). In Campylobacter spp., the genetic locus encoding the flagellin is formed by the flaA and flaB genes arranged in tandem. This locus has both variable and highly conserved regions, which make it suitable for molecular typing [8, 11]. The RFLP methodology exploring the polymorphisms within the flagellin locus is fairly inexpensive, is relatively simple to perform, and is well suited for relatively high-throughput analysis. Still, some limitations have been reported over the years, especially the possibility for genetic recombination in the flagellin locus [12], which hamper molecular tracking in epidemiologically related strains. This method is also not appropriate for global and long-term duration epidemiological studies as a single approach. Over the years, it has been progressively replaced, or complemented, by other methodologies with higher discriminatory power and reproducibility, such as multilocus sequence typing (MLST), based on the nucleotide sequence analysis of housekeeping genes, and the long-standing gold-standard technique, pulsed-field gel electrophoresis (PFGE). On the other hand, flaA-RFLP analysis can be very useful in epidemiological studies limited in space and time (e.g., to genotype the isolates from poultry flocks) and, for specific epidemiological scenarios, an alternative to the more resource-demanding techniques mentioned above. Its low cost associated with the quick procedures of the workflow also stands out as major advantage. This method was recently applied with success to the comparative genotypic analysis of C. jejuni and C. coli isolated from broilers in a nationwide survey in Portugal [13].
Several experimental protocols have been developed for Campylobacter spp. typing based on the amplification and restriction of the flaA gene, which differ considerably in DNA preparation, the set of primers, the annealing temperature used in PCR, and the restriction enzymes [8]. Various restriction enzymes have been used individually or in combination, in particular, AluI, DdeI, HinfI, EcoRI, and PstI [14]. The methodology reported in this work is based on the procedure proposed by the Campynet network, which in turn is based on the method originally described by Nachamkin and coworkers [15, 16]. Here, we complement the former protocols with a few adaptations that have been developed to improve the reproducibility and accuracy of the flaA-RFLP patterns. From the experimental viewpoint, flaA-RFLP analysis can be subdivided into five major procedures: DNA extraction, amplification of a fragment of the flaA gene by PCR, amplicon restriction with different endonucleases, electrophoretic separation of generated fragments, and RFLP analysis using suitable software.
2 Materials
2.1 Growth of Campylobacter spp. and Maintenance
1.
Growth media: Prepare Columbia blood agar base according to manufacturer’s instructions. Sterilize by autoclaving at 121 °C for 15 min. Cool to 50 °C and aseptically add 5 % (v/v) of defibrinated lysed horse or sheep blood.
2.
Cryopreservation media: Prepare a mixture of 40 % (v/v) glycerol and 6 % (w/v) Tryptone Soya Broth.
3.
Incubation jars of 2.5 L or special incubation bags for the incubation of Petri dishes in an oxygen-depleted and CO2-enriched atmosphere (see Note 1 ).
4.
Microaerophilic gas generator systems suitable for incubation bars or for incubation bags.
5.
Incubator at 42 °C.
2.2 Isolation of Genomic DNA
Prepare all solutions using ultrapure water. Purify deionized water to reach a resistivity of 18.2 MΩ cm at 25 °C. Keep the reagents/buffers at room temperature, unless otherwise indicated.
1.
SET buffer: 150 mM NaCl, 15 mM EDTA, 10 mM Tris–HCl, and pH 8.0.
2.
Sodium dodecyl sulfate (SDS) in distilled, deionized water: Prepare a 10 % (w/v) stock solution (see Note 2 ).
3.
Proteinase K: Prepare a stock solution of 20 mg/mL in distilled water. Keep it at −20 °C.
4.
Phenol/chloroform/isoamyl alcohol (25:24:1) solution, saturated with 10 mM Tris (pH 8.0) and 1 mM EDTA. Keep it at 4 °C.
5.
3 M sodium acetate; pH 5.3.
6.
Absolute and 75 % (v/v) ethanol.
7.
Sterile 1 mL syringes.
8.
1.5 mL sterile tubes.
9.
Benchtop centrifuge.
10.
DNA vacuum dryer equipment.
2.3 Amplification of flaA Gene by Polymerase Chain Reaction (PCR)
1.
Sterile PCR microtubes.
2.
Reagents for conventional PCR reactions: Taq DNA polymerase and the respective 10× reaction buffer, 25 mM MgCl2, 10 mM deoxynucleoside triphosphate mix (dNTPs), and 0.1 mM of each oligonucleotide primer (primer A1: 5′-GGA TTT CGT ATT AAC ACA AAT GGT GC-3′ and primer A2: 5′-CTG TAG TAA TCT TAA AAC ATT TTG-3′) [15]. Commercially synthetized primers are lyophilized and need to be diluted with nuclease-free water. Stock solutions can be prepared at a standard concentration of 100 pmol/μl and stored at −20 °C. To avoid freeze and thaw, aliquots of 10 pmol/μl working solutions of each primer may be prepared from stock solutions in water and stored also at −20 °C.
3.
Sterile PCR-grade nuclease-free water.
4.
Reference strains as controls: C. jejuni subsp. jejuni ATCC 33560 and C. coli CCUG 11283.
5.
Standard PCR equipment (thermocycler).
2.4 Agarose Gel Preparation and Electrophoresis
1.
5× TBE buffer: 0.89 M Tris Base, 0.89 M boric acid, and 0.5 M EDTA; adjust pH to 8.0. Dilute 200 mL of 5× TBE buffer in 800 mL of purified deionized water to obtain 1 L of 1× TBE buffer. Store at room temperature.
2.
Agarose gel: In a glass bottle (see Note 3 ), add the appropriate amount of agarose (electrophoresis grade) to 100 mL of 1× TBE buffer [e.g., 1 % (w/v) agarose gel contains 1 g of agarose]. Put the bottle with the cap unscrewed in a microwave (used only for this purpose). Heat until completely liquefied (about 3 min). Let it cool down to 50 °C. Add ethidium bromide at 0.3 μg/mL to the gel (see Note 4 ). Ethidium bromide is a known mutagen and should be handled as a hazardous substance. Put the gel into a suitable mold (midi gel dimensions: about 12 cm wide by 10 cm long; pour 100 mL of molten volume). Put one comb inside the mold before the gel polymerizes. After 30/40 min, the gel should be ready to use (see Note 5 ). Remove the combs and put the mold with the gel in an electrophoresis tank. Use 1× TBE as running buffer. The gel must be completely covered by the buffer.
3.
Molecular weight markers: Select up to three ladders producing regularly spaced 100 bp, 50 bp, and 25 pb fragments.
