Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.


Introduction
Since its initial recognition in the 1970s, Campylobacter has become one of the most common causative agents of bacterial foodborne gastroenteritis in humans [1,2]. The incidence of human Campylobacter infections is increasing worldwide along with the proportion of isolates resistant to antibiotics [3]. Animals, including sheep and goats, are the natural reservoir hosts for Campylobacter spp. Although most infections in small ruminants are asymptomatic, there is recent recognition of a zoonotic clone, sequence type-(ST)-8, which is associated with ovine abortions in a number of states within the United States [4,5]. Recent studies report a wide range (5% to 49%) of Campylobacter prevalence in healthy sheep and goats from different geographic areas [6][7][8][9]. Several studies conducted in high sheep and goat producing areas indicate that these small ruminants may be important sources of human exposure [3,[6][7][8][9][10][11][12][13][14][15].
Grenada is a small, developing island state in the Caribbean where many residents are involved in small-scale sheep and goat production for meat, milk, and milk products. It is likely that, in a predominantly rural country like Grenada, sheep and goats serve as important sources for human exposure by direct contact or via environmental or food exposure. The goal of this study was to determine whether sheep and goats are a reservoir host for pathogenic and antibioticresistant Campylobacter spp. Another goal was to characterize Campylobacter population structure among these animals by multilocus sequence type (MLST) genotyping of the isolates. 2 Veterinary Medicine International and Carriacou, islands in the tri-island state of Grenada, were obtained between May and July, 2011. The much smaller island of Carriacou is similar in land mass and population to the individual parishes in Grenada [16] and thus, for the purposes of this study, the island of Carriacou was considered as an island consisting of one parish. Based on the Grenada Ministry of Agriculture, Forestry and Fisheries report on the number of sheep and goats inspected in Grenada [17], we estimated a population size of 3000 sheep and 2500 goats on these 2 islands at the time of our sampling. Given these population estimates we sampled greater than 5% of the sheep and greater than 10% of the goats on these two islands. At the 95% confidence level our sample sizes were sufficient to detect a 5% fecal shedding rate with confidence intervals of ±3.34% and ±2.55% in sheep and goats, respectively (http://www.surveysystem.com/sscalc.htm).
Groups of sheep and/or goats owned by one person or family were defined as a production unit. Twenty-one production units were selected from the 6 parishes of Grenada and 8 production units were selected from the sister island of Carriacou. Production units ranged in size from 10 to 120 animals. Approximately 25% of the sheep and goats within these production units were sampled. Samples were from clinically healthy animals that were at least 6 months old. Health status was determined by physical exam by a veterinarian. Culture swabs containing Cary-Blair transport medium (BBL, Becton, Dickinson and Company, Sparks, MD, USA) were used for sample collection. Samples were transported on ice to the St George's University School of Veterinary Medicine Diagnostic and Research Laboratory for processing the same day.

Bacterial Culture and Identification.
Rectal swabs were plated onto Campylobacter blood-free selective agar with cefoperazone and amphotericin B supplements (Modified CCDA-Preston plates, Oxoid Ltd., Basingstoke, UK). The plates were incubated under microaerophilic conditions at 42 ∘ C for 48 h using a microair atmosphere generating system (GENbox microaer system, 96126 BioMerieux UK Ltd, Basingstoke UK). Presumptive Campylobacter colonies were identified by their typical grey-white, mucoid flat appearance. Campylobacter morphology was confirmed by Gram staining. Growths of pure cultures were transferred into 2% sterile skim milk in cryovials and stored at −85 ∘ C. To speciate the isolates, the hippurate test was used as previously described [18]. A multiplex PCR that differentiates between C. jejuni, C. coli, C. lari, and C. upsaliensis on the basis of amplicon size of the IpxA gene was also used [19].

Antimicrobial Susceptibility
Testing. Antimicrobial susceptibility/resistance of Campylobacter isolates ( = 15) was determined for the following seven antibiotics: gentamicin, chloramphenicol, ciprofloxacin, ampicillin, tetracycline, erythromycin, and metronidazole. Susceptibility and resistance to metronidazole has been proposed as an epidemiological marker for C. jejuni of avian origin [20] and may have relevance to ruminant-associated C. jejuni as well. The other six antibiotics are all used to treat human cases of campylobacteriosis. The Epsilometer test (E-test, AB Biodisk, Solna, Sweden) for minimum inhibitory concentration (MIC) was conducted according to the manufacturer's instructions on Mueller Hinton agar (Remel, Lennexa, KS, USA) as previously described [18]. A C. jejuni strain of poultry origin susceptible to all seven drugs and giving reproducible MICs was used as a control. The MIC of a drug was read directly from the scale printed on the E-test strip at the point of intersection between the zone of bacterial growth and the rest of the strip. Break points used by Guévremont et al. [21] and those established by the Clinical and Laboratory Standards Institute [22] for the microbroth dilution test on aerobic bacteria were used to interpret MICs. The break point for resistance to metronidazole was set at ≥16 g mL −1 , as recommended by Lorian [23]. MIC values used to classify a strain as resistant were ampicillin ≥32 g mL −1 , chloramphenicol ≥32 g mL −1 , ciprofloxacin ≥4 g mL −1 , erythromycin ≥8 g mL −1 , gentamicin ≥16 g mL −1 , tetracycline ≥16 g mL −1 , and metronidazole ≥16 g mL −1 .

Multilocus Sequence Typing (MLST).
Stock cultures of the 7 isolates from sheep and 8 isolates from goats were grown on blood agar plates for 24 h at 42 ∘ C under microaerophilic conditions. Total DNA was extracted using DNA blood and tissue purification kits (DNeasy Blood & Tissue Kits, Qiagen Inc., Valencia, CA, USA) according to manufacturer's instructions and samples were shipped to the College of Veterinary Medicine, University of Minnesota, St. Paul, MN, for MLST determination. PCR amplification of seven genes was carried out as described previously [24][25][26] using guidelines and PCR primer sequences obtained from the Campylobacter PubMLST database (http://pubmlst.org/campylobacter/mlst-info/Cjejuni/primers.html). Briefly, PCR was performed using commercial ready-to-use master mixes in a master cycler (Mastercycler (Eppendorf AG), HotStarTaq Master Mix kit, Qiagen Inc., Valencia, CA, USA). The reaction mixture contained 10 M of each primer, 25 L of PCR master mix, and 2 L of template DNA and water to make total volume of 50 L. The cycling program consisted of an initial denaturation at 95 ∘ C for 15 min, followed by 35 cycles of 94 ∘ C for 30 sec, respective annealing temperature for each primer for 1 min, and extension at 72 ∘ C for 3 min with a final extension at 72 ∘ C for 10 min. To confirm the presence of amplicon of the expected size, the PCR amplification products were run on 1.2% agarose gel. Purified PCR products were quantified and sequenced (ACGT, Inc., Wheeling, IL, USA) using the same primers used for the PCR amplification. The obtained sequences were analyzed using sequence analysis software (Sequencherver 10.1, Gene Codes Corporation, MI, USA). Sequence data were submitted to the Campylobacter PubMLST database for allele assignments.

Data Analysis.
A two-tailed Fischer's Exact test was used to compare the percent of sheep and goats positive for fecal Campylobacter, prevalence rates among parishes, and the percent of isolates resistant to the various antibiotics. Veterinary Medicine International 3 A value of ≤ 0.05 was considered significant. Sequence analysis and determination of sequence types (ST) and clonal complex (CC) were performed with the integrated network MLST application for Campylobacter (SmartGene Inc, Raleigh, North Carolina, USA) which uses the PubMLST database http://pubmlst.org/campylobacter/ for ST and CC designation. A neighbor-joining tree based on allelic sequences was constructed using a suite of analysis programs (PHYLIP analysis programs) available on the PubMLST website http://pubmlst.org/analysis/. Tree drawing was performed using PubMLST Phylodendron software also available at the same PubMLST website. Tree branches are labeled by animal source (sheep or goat) and the corresponding ST assignment.
Isolates not yet assigned to an ST are labeled as "Novel. ''

Campylobacter Species. Fifteen
Campylobacter spp. isolates were obtained from the 407 animals sampled ( Table 1). All isolates were phenotypically typed by the hippurate test as either C. jejuni or C. coli, with 14 of the 15 isolates (93.3%) identified as C. jejuni (6 from sheep and 8 from goats) and 1 (6.7%) of the isolates identified as C. coli (from a sheep on the island of Carriacou). Results of speciation were further confirmed by multiplex PCR. The overall prevalence of Campylobacter fecal shedding was 3.7% and did not differ significantly between sheep (4.5%) and goats (3.2%) ( = 0.59). The sheep/goat prevalence ranged from 0% in the parish of St. George to 20% in the parish of St. Patrick. These were the only two parishes that differed significantly from each other for percent positive animals ( = 0.0019).

MLST Profiles and Antibiotic
Resistance. MLST genotyping was completed on 13 of the 15 isolates (Table 2). For the other 2 isolates neither CC nor ST determinations were possible because amplification products were not detected for one or more loci. Although PCR conditions were optimized and sequencing was repeated, additional primers were not used to fill the missing gaps for ST designation due to limitation of sample. Five CCs identified in this study have previously been described ( Eight of the 15 Campylobacter isolates were resistant to either metronidazole or tetracycline or both (Table 3). There was no significant difference between the percent of isolates resistant to tetracycline (30.8%) and the percent resistant to metronidazole (38.5%) ( = 1.0). All of the metronidazoleresistant isolates were from Carriacou. This included 3 of the goat C. jejuni isolates and both of the C. coli isolates from sheep. Tetracycline-resistant isolates were from Carriacou, St. Patrick, and St. David and included the C. coli metronidazoleresistant Carriacou sheep isolate. All four of the tetracycline resistant isolates belonged to the ST-21 clonal complex and encompassed 3 of the 4 ST-21 clones.
A cladogram constructed from the 13 typable Campylobacter MLST allelic profiles illustrates the clustering of the STs identified in this study and demonstrates the genetic relatedness of sheep and goat isolates and the lack of any shared STs between sheep and goats ( Figure 1).

Discussion
The aims of this study were to determine the prevalence, genetic diversity, and antibiotic resistance of Campylobacter isolates from sheep and goats in Grenada. The Campylobacter prevalence in sheep (4.5%) and goats (3.2%) in this study is generally lower than that reported from other geographic  areas [6,7,9,12,15]. However, similar to these reports and the PubMLST data base, the majority of our isolates were C. jejuni. C. coli was isolated from only one sheep from the island of Carriacou. Because this isolate was not MLST typable, one must consider the possibility that the isolate was not a Campylobacter. The low prevalence results from our study may reflect sample size, sampling biases, or the lack of Campylobacter MLSTs in Grenada that predominantly colonize sheep/goats. The lower prevalence noted in this study may also be due to the fact that the animals in Grenada are kept in a free range system and there is no intensive farming on the island. Enrichment methods were not used because, compared to direct plating, they were shown to be inefficient for isolation of Campylobacters from fecal samples from animals [27][28][29]. Musgrove et al. [30] noted that large numbers of non-Campylobacter species that inhabit the intestinal tract may outcompete Campylobacters during enrichment, confounding detection. However, information on enrichment techniques with regard to fecal sample of sheep and goats is lacking, and further research may elucidate this aspect. Most of the ST clones identified in our study are STs that are globally associated with poultry as a host species (Campylobacter PubMLST Database, 2012). Regardless, C. jejuni and possibly C. coli are present in small ruminants in Grenada.
In this study we identified eleven ST clones including eight previously reported STs within five recognized CCs and three novel clones not yet assigned. Of major interest is the detection of the previously reported clone ST-8 in CC ST-21 in a single goat in our study. It was recently reported that the vast majority of C. jejuni isolates associated with sheep abortion in several US states belonged to this single ST-8 clone, referred to as clone SA for sheep abortion. Further, as with the isolate from the goat in this study, all were resistant to tetracycline, the only antibiotic class currently approved in the USA for treatment of Campylobacter abortion in sheep [4]. More recently clone SA was reported in association with human disease [5]. Although the PubMLST database only shows one report of ST-8 in a goat, other goat ST-8 isolates have been reported in association with abortions [5]. Because not all ST-8 isolates may be the SA clone, additional testing of our ST-8 isolate would be required to determine if it is indeed identical to the SA clone. Because sheep or goat abortion cases in Grenada are rarely seen by a veterinarian The major lineages identified in this study correspond to CCs that are primarily associated with sporadic human disease and poultry and are geographically widely distributed (Table 4). However, none of the sheep/goat isolates from this study correspond to any of the STs previously identified in poultry in Grenada [18]. Because of the type of agriculture practiced in Grenada where biosecurity between various animal production units is minimal and comingling of species is common, these observations support host species-associated Campylobacter STs due to factors other than host species containment. This observation is similar to results from a study in Scotland of sheep and cattle exposed to the same farming environment, but that consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs [31].
The Campylobacter isolates ( = 15) genotyped into 11 different STs, indicating considerable population diversity in both sheep and goats. Four of the 8 previously reported STs identified in this study (ST-21 sequence type 454, ST-21 sequence type 5340, ST-677 sequence type 5020, and ST-353 sequent type 5908) have never been reported from sheep before. Five of the 8 previously reported STs identified in this study have never been reported in goats before, with only ST-21 sequence type 50 and sequence type 8 being previously reported. Most of our isolates represent STs reported from the UK, The Netherlands, Germany, and Canada ( Table 4). Two of the STs from our study have previously been reported from the Caribbean island of Curacao, all 12 of which were from human cases of gastroenteritis (Campylobacter PubMLST Database, 2012).
CC ST-21 was the dominant CC for both sheep and goat isolates ( Table 2) and is represented globally and from multiple host species including sheep and goats (Table 4). 6 Veterinary Medicine International The Campylobacter isolates showed no significant difference between sheep and goats for either the type of antibiotic resistance or the percent of antibiotic resistant isolates (Table 3). Resistance was only detected for tetracycline and metronidazole. The level of Campylobacter tetracycline resistance in this study (57% of ovine isolates and 50% of caprine isolates) is in close agreement with those from Canada [32], Spain [33], and Iran [8,12]. The E-test results are known to correlate well with agar dilution and broth dilution methods for erythromycin and ciprofloxacin. However, it has been noted that the overall agreement between E-test and broth microdilution method is only 90%, and even less with agar dilution methods [34,35].
Results from this study contribute to the understanding of Campylobacter population structure within and among host species in Grenada. Sheep and goat rearing in this island state consists of small production units which are often in close association with back yard or small-scale poultry and swine production. This type of animal agriculture is common in many parts of the world and findings from Grenada may shed light on Campylobacter population structure, spread, and antimicrobial resistance relevant to many geographic areas.

Conclusions
This report of genetic typing of Campylobacter isolates from sheep and goats is the first in the Eastern Caribbean. Results from this study document that both sheep and goats are reservoirs for antibiotic resistant Campylobacter jejuni/coli. Results also document that the previously identified, highly pathogenic Campylobacter jejuni genetic clone for ovine abortion in the USA, ST-8, is also present in Grenada. Identical to the US isolates, the ST-8 isolate from a goat in Grenada is also tetracycline-resistant. Campylobacter isolates in this study are primarily from comingled sheep and goat production units and yet no shared genetic clones were identified between these two host species. None of the sequence types identified in this study match those previously reported Veterinary Medicine International 7 in poultry in Grenada, suggesting the presence of sheep-and goat-specific Campylobacter clones in Grenada.