3.4.1. Study design
This was a prospective cross-sectional study performed between February and July 2010 using a convenience sample of client-owned dogs and cats examined at the Veterinary Teaching Hospital at Massey University, Palmerston North, New Zealand. Only animals examined for routine vaccination, neutering, health check, and for dental treatment were included in the study. Rectal swabs were taken and information was obtained at the time of sampling regarding age (categorised as young or adult at a cut-off of six months), sex, breed, vaccination and deworming history, neuter status, diet fed (including raw meat), signs of illnesses and any recent drug treatments, the presence of other animals in contact with the case, shelter/boarding kennel history and hunting behaviour. The study was independently reviewed and approved by the Massey University Animal Ethics Committee under application number 09/70. Concurrent to sampling of pets, 50 raw pet food products were purchased at five commercial outlets located in Palmerston North. Food products were arbitrarily selected and the sellers were unaware of the intended use.
3.4.2. Bacterial culture
All rectal swabs were transported without refrigeration using Amies charcoal transport medium (Copan, Brescia, Italy) due to the close proximity of the on-site laboratory and were cultured within 2 hours of sampling using two protocols: (i) direct plating onto CAT agar (Fort Richard, Auckland, New Zealand) with incubation at 37°C in an envelope-generated (CampyGen, Oxoid Ltd., Basingstoke, UK)
microaerobic atmosphere in gas-jars and (ii) 48 hours enrichment in 3mL of Bolton Broth (Lab M, Bury, UK) with subsequent plating onto mCCDA plates (Fort Richard, Auckland, New Zealand) at 42°C under microaerobic conditions (85% N2, 10% CO2,
5% O2) using a variable atmosphere incubator (Don Whitley Scientific, West
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day of incubation, whilst mCCDA plates were only checked on the fourth day. All fresh raw pet food samples were cultured within two hours of purchase and frozen samples were first thawed at room temperature before culturing. Approximately 10g of the food samples were aseptically removed from the packaging and briefly mixed in a stomacher with 90mL of Bolton Broth which was split in two bottles for enrichment of 48 hours at 37°C and 42°C followed by plating onto mCCDA and CAT
plates for another 48 hours; all under conditions as described above for the respective plates. Control plates were used for all incubation conditions. Colonies with morphological features indicative of Campylobacter spp. were checked for size and motility by dark-field microscopy. Two individual colonies (as available) each of different morphology indicative of Campylobacter spp. by dark-field microscopy were subcultured using Columbia horse blood agar (Fort Richard, Auckland, New Zealand). Whole plates of pure colonies were harvested for storage in 15% (weight/volume) glycerol in nutrient broth vials (Difco Laboratories Inc., Franklin Lakes, NJ, USA) at -80°C. Isolates negative by the PCR identification protocol below
were regrown and tested for growth at room temperature as a phenotypic test to exclude Campylobacter spp. status, and if aerotolerant and non-thermophilic, were further tested by a PCR specific for Arcobacter butzleri.
3.4.3. Isolate identification and genotyping
Crude DNA extraction was performed by boiling fresh cultures for 10 min in an aqueous 2% (volume/volume) Chelex® solution (Bio-Rad Laboratories Inc., Hercules, CA, USA) followed by centrifugation and decantation of the supernatant into a sterile tube and storage at −20°C until each PCR was run. Each sample was initially tested by C. jejuni PCR and if negative, by a Campylobacter genus PCR. Subsequent to a positive genus PCR, samples were sequentially tested by species- specific PCRs that targeted C. coli, C. upsaliensis, C. helveticus, C. lari, C. fetus or C. hyointestinalis. In addition, A. butzleri PCR and 16S rRNA gene sequencing were performed for a subset of isolates negative by all species-specific PCR that exhibited growth at room temperature and room atmosphere. The detailed description of primers, amplification protocols, and references are presented in Supplemental Table
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3.1. Controls were used for all PCR assays and amplified products were identified by electrophoresis in a 1% (weight/volume) agarose gel in TBE buffer (along with a 1Kb Plus ladder (Invitrogen Corp., Waltham, MA, USA)), subsequently stained with ethidium bromide and exposed to UV light using a Bio-Rad gel documentation system (Olympus Life Science Group, Richmond Hill, Ontario, Canada). Isolates confirmed as C. jejuni by PCR were genotyped using the Campylobacter-specific multilocus sequence typing (MLST) scheme of seven housekeeping genes as described previously (256). The amplifications were performed in a 25μL volume reaction using Applied Biosystems AmpliTaq Gold mastermix (Applied Biosystems, Auckland, New Zealand) and 5 pmol of each primer. Products were sequenced on an ABI 3130XL automated DNA sequencer using ABI BigDye v3.1 (Applied Biosystems, Auckland, New Zealand) following the manufacturer's instructions. Sequence data were collated and alleles assigned using the Campylobacter PubMLST database (http://pubmlst.org/campylobacter/) and sequence types (ST) compared with those contained in the mEpiLab database. The mEpiLab database contained over 3,500
samples (at the time of writing) from the Manawatu Campylobacter sentinel surveillance site, a ten year plus project for source attribution of campylobacteriosis using concurrent sampling of sick people, animals, food and the environment (260).
3.4.4. Statistical analysis
The power analysis performed using G*Power v3.1 (456) for sampling of a total of 200 animals and 50 food samples with a two-tailed binomial test (α = 0.05, β = 0.1) showed the effect size of 0.17 for 100 samples each from dogs and cats and for 50 samples (for raw food) the effect size of 0.23 could be detected. Isolates positive by PCR were used to report an apparent prevalence and 95% CI based on a binomial distribution using the Clopper and Pearson method (457). Univariate analyses to test the association of Campylobacter status with collected animal/food information were performed using Fisher’s exact test of independence. Multivariate logistic regression analysis was performed using the least absolute shrinkage and selection operator (LASSO) regression. All variables with p < 0.3 from the univariate analysis were used for LASSO profiling by the lambda penalty parameter and cross-validation of the
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model log-likelihood. The number of non-zero coefficients and 95% CIs were estimated using 10,000 bootstrapped replicates of equivalent sample sizes with replacement. Culture methods were compared using Fisher’s exact test of independence and unweighted Cohen’s kappa as an index of interrater agreement. The New Zealand National Dog Database (www.localcouncils.govt.nz) reports were used for the evaluation of the sampled dog population profile. Statistical and exploratory data analyses were performed using R v3.2.2 (R: A language and environment for statistical computing. R Core Team (2013). R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/).