The aim of the study was to identify the isolation rate of thermotolerant campylobacters in a small-scale broiler-meat production farm over a one-year period. The second deliverable of the study was to determine the potential virulence markers. The laboratory investigation was performed on 283 samples (cloacal swabs, caeca, carcass swabs) collected on three sampling points (farm, slaughter line, and cold storage). The isolates obtained with the conventional microbiological method were confirmed with multiplex PCR for identification of campylobacters. The presence of 10 virulence genes was analyzed in the C. jejuni isolates ( flaA, racR, virB11, dnaJ, wlaN, cadF, ciaB, cdtA, cdtB, cdtC). Out of 283 samples, 169 (59.7%) were confirmed as Campylobacter spp., 111 (39.2%) C. jejuni, and 43 (15.2%) C. coli. C. jejuni was the most prevalent in all sampling points. Campylobacter spp. showed a characteristically seasonal prevalence with the highest isolation rate during the warmer period of the year. We detected the cadF and ciaB genes in all C. jejuni isolates. The flaA gene was present in 50% of the examined strains. The cdt genes (cdtA, cdtB, and cdtC) were confirmed in 52.8%, 52.8%, and 47.2% of the C. jejuni strains, respectively. C. jejuni showed 15 profiles of virulence patterns with four predominant profiles.
is the most commonly reported causative agent for human bacterial diarrhoeal disease in the European Union since 2005 (5
). The main source of infection is by consumption of food with animal origin, especially undercooked poultry meat, unpasteurized milk, or cross-contaminated meat products during preparation and storage (6
). Besides these risk factors there are several more that are worth considering: abroad traveling, exposure to animals, working in slaughterhouses, animal farms visiting, consumption of pork meat, contact with dogs and cats, and swimming in open waters (7, 8, 9, 10
The infection is usually brief, mild, self-limiting, and generally does not require any treatment. In some cases, the infection is associated with serious clinical manifestations such as bacteremia, reactive arthritis, hemolytic uremic syndrome, meningitis, septicemia, and Guillain-Barré syndrome (1, 11
). Macrolides and fluoroquinolones are frequently used as the first and second-choice drugs for highrisk patients (young, elderly, immunocompromised individuals) (12
Many authors have confirmed the seasonal pattern variation in the occurrence rate of Campylobacter
. The usual prevalence spikes take place during the warmer months of the year and are generally associated with the higher survival of Campylobacter
in the habitat (13
The molecular pathogenic mechanism in Campylobacter
infection is still not perfectly understood (14
). The extensive research on campylobacters has revealed numerous genes relevant for their virulence and pathogenicity (15
): flaA, flhA, cadF, dnaJ,
(genes that are engaged in the stage of adherence and colonization); virB11
, and iam
(genes important for the invasiveness); cdtA, cdtB,
(genes bound to the cytotoxic effect on the epithelial cells), and wlaN
(linked with the synthesis of the lipooligosaccharides, resulting in Guillain-Barré Syndrome) (16
The objectives of this study were to 1) estimate the isolation rate of thermotolerant Campylobacter
in broilers sampled in farms and slaughterhouse; 2) identify (if present) the seasonal variation in Campylobacter
prevalence; 3) detect the presence of virulence-related genes in Campylobacter jejuni
isolates, and 4) evaluate the detected virulence patterns in Campylobacter jejuni
Collection of samples
MATERIAL AND METHODS
This study was conducted on one broiler farm and slaughterhouse in the Skopje region, N. Macedonia, in the period between March and December 2017. All procedures involving animals were in accordance with the ethical standards and international guidelines for the care and use of animals.
A total of 283 samples were collected. The farm had a capacity of 10.000 broilers with a conventional system. The usual rate of slaughtering was by several thinning cycles of 1.500 broilers per day.
Cloacal swabs were taken from randomly chosen broilers at the farm one week prior to slaughter (n=64). The samples were placed in plastic tubes containing 5 mL of Preston broth.
Broiler caeca were collected at the slaughter line (n=166) during the evisceration phase, and were packed in sterile plastic bags. Samples were collected randomly from at least 40 birds in each sample-collection cycle during the slaughtering process.
Swab samples from broiler carcasses (n=53) were taken in the storage area before shipping to the consumers (cold chamber of the slaughterhouse) using sterile cotton swabs, which were placed in plastic tubes containing 5 mL of Preston broth. The samples were collected the following day after slaughtering, including at least 10 carcasses in each sample-collection cycle.
Samples were stored and transported to the laboratory at 4-8 °C. The laboratory analysis was initiated 3-4 hours after sampling. Isolation and confirmation of Campylobacter spp.
The isolation and identification of thermotolerant Campylobacter
was performed according to the standard ISO 10272-1:2017 (17
). The positive isolates were sub-cultured on mCCDA agar plates and stored in glycerol broth at -80 °C.Extraction of DNA and PCR analysis
The DNA preparation for the Campylobacter
isolates was performed by the boiling method. The procedure included suspension of the cultures in 0.5 mL of TE buffer, boiling at 95 °C for 10 min, and centrifugation at 15.000 rpm for 5 minutes. Acquired supernatants were kept at -20 °C and used for both PCR methods.
The multiplex PCR for identification and differentiation of Campylobacter
spp. was performed with adaptations according to the previously published study (18
). This multiplex PCR could identify several Campylobacter
species (C. jejuni subsp. jejuni
; C. coli, C. lari, C. upsaliensis
, and C. fetus subsp. fetus
). The list of the used primers is shown in Table 1
PCR amplifications were performed in a mixture (25 μL) consisting of 12.5 μL of 2× Platinum Multiplex PCR Master Mix (Applied Biosystems, UK), 2.5 μL of template DNA, and 5 μL of primer mix (0.5 μM C. jejuni
and C. lari
primers; 1 μM C. coli
and C. fetus
primers, 2 μM C. upsaliensis
primers; 0.25 μM 23S rRNA primer). Distilled water was added to make 25 μL. DNA amplification was carried out in a thermocycler (Techne, UK) using an initial denaturation step at 95 °C for 2 min followed by 35 cycles of amplification (denaturation at 95 °C for 30 sec., annealing at 59 °C for 45 sec., and extension at 72 °C for 45 sec.), ending with a final extension at 72 °C for 10 min.
The multiplex PCR for detection of the virulence genes in the C. jejuni
isolates was performed as proposed by Datta (19). The list of the used primers is shown in Table 2
For this protocol each multiplex PCR tube contained 25 μL of mixture: 12.5 μL Platinum Multiplex PCR Master Mix (Applied Biosystems, UK), 2.5 μL of template DNA, 5 μL of primer mix (0.5 μM of the used primers), and 5 μL of distilled water. Amplification was performed with initial denaturation step at 95 °C for 2 min followed by 35 cycles of amplification (denaturation at 94 °C for 1 min, annealing at a temperature specific to the primer pair for 1 min, and extension at 72 °C for 1 min) and final extension at 72 °C for 10 min.
Isolation rate of thermotolerant Campylobacter, C. jejuni, and C. coli at broiler farm and at the slaughterhouse
The results of the study indicated that Campylobacter
spp. was present in all phases of the production. The distribution of campylobacters was highest in the cloacal swabs (73.4%). The results of the laboratory examinations showed that C. jejuni
was the most prevalent species on the farm and in the slaughterhouse (Table 3
This study identified high prevalence of Campylobacte
r in carcasses (37.7%), consisting mainly of isolates of C. jejuni
(22.6%), followed by C. coli
(9.4%).Seasonal variation in Campylobacter spp. prevalence
As previously mentioned, in several studies, Campylobacter
spp. has shown a seasonal trend of higher prevalence in the warmer period of the year. In the current study (Fig. 1
) we confirmed this trend with highest prevalence in the samples taken in August (75.3%), followed by the June sampling period (67.1%).Figure 1.
Seasonal prevalence of Campylobacter
spp. along the broiler chainPresence of virulence genes in C. jejuni
Among the isolates, 111 were confirmed as C. jejuni
by both the classical method and the multiplex PCR. Three of the isolates were dismissed because of technical issues with the template DNA purity. Therefore, 108 isolates were subjected to further analysis for the detection of virulence genes (Fig. 2
; Table 4
Visualization of PCR amplicons of virulence genes. Lane marked with M: 100-bp ladder;
Lane 1: racR, dnaJ
genes; Lane 2: cadF, racR
genes; Lane 3: cdtC, cdtA
genesEvaluation of the detected virulence patterns in C. jejuni
In total, we detected 15 virulence patterns in the C. jejuni
isolates. In 19.4% of the confirmed C. jejuni
isolates, the analysis detected the profile No. 7. The second highest prevalence was detected for the profile No. 12 (in 13.9% of the isolates). The profile No. 1 (all of the virulence genes present except virB11
) was confirmed only in 3 isolates (2.8%) of C. jejuni
In this study, Campylobacter
spp. was present in all type of samples collected along the broiler meat production chain (cloacal swabs, caeca, and carcass swabs). The confirmed level of Campylobacter
spp. on the broiler farm was very similar with the level confirmed in two studies performed on farms with comparable breeding conditions (20, 21
This study also revealed high isolation rate of Campylobacter
spp. (61.4%) in the slaughterhouse (evisceration phase) in the cecum samples. Analysis of cecal samples showed a great variation in the isolation rate (region, age of the flock, seasonality, on farm pre-harvest measures, farm management) (22
). In some studies, the isolation rate was in accordance with our results (23
), but there are studies that report even higher isolation rate (24
At the last point of sampling (cold storage), 37.7% of the samples were positive. The point of concern is that 22.2% of the samples have shown to be positive for C. jejuni
. This is important as from this point the poultry meat is dispatched to the consumers. If we follow the Campylobacter
spp. presence along the production chain, we find reduced level but not eliminated level of contamination (25
Our results have confirmed a well-defined seasonal pattern of flock colonization with Campylobacter
spp. As observed by many authors, it is characterized with highest prevalence in summer or autumn (26, 27, 28
). The mechanism by which temperature affects Campylobacter
colonization of broilers is probably linked with microbial survival, higher numbers of wildlife vectors, increased ventilation and fan speeds, insects, and rodents (29
The pathogenicity of thermotolerant Campylobacter
found in the broiler meat in addition to its isolation rate gives a clear picture of the public health risk. In this study we detected several of the virulence genes in C. jejuni
isolates. Our results showed that cadF
genes were detected in all of the confirmed strains (Table 5
). Similar observations indicated that the cadF
genes were present in the Campylobacter
isolates from chicken carcasses and droppings (19, 30
The analyses detected an isolation rate of 50% of the flaA
gene which coordinate the motility and virulence (31, 32
). Comparable levels of prevalence of this gene were detected in other studies (33, 34
The frequency of cdt
genes (52.8%, 52.8%, and 47.2%) that was observed in our C. jejuni
isolates was close to the prevalence detected in studies by other authors (35, 36
). This holotoxin (cdtABC) produced by campylobacters is a factor which has a great role in the development of the disease. All three toxin subunit genes (cdtA, cdtB
, and cdtC
) are necessary for the cytotoxin expression (37
gene (prevalence of 13.9%) is responsible for the metabolism of specific lipooligosaccharides (LOS) connected with disease sequelae such as Guillain-Barre syndrome (38, 39
). Concerning the genes connected with adherence and colonization (40
) the study results confirmed 44.4% prevalence of the dnaJ
gene and lower prevalence of the racR
gene (27.8%). The virB11
gene, which is responsible for the expression of invasion (41
), was not detected in the C. jejuni
The authors acknowledge that at the time of sample collection for the purpose of the current research, the country had a small-scale broiler production based on few farms and only one operating slaughterhouse imposing limitations of sample number.
This study confirmed high isolation rate of Campylobacter
spp. in the broiler meat production chain. This statement is supported both at the farm and the slaughterhouse (evisceration phase and cold storage) in different type of analyzed samples. C. jejuni
was more frequently isolated than C. coli
at the three sampling points.
isolation rate in the cold chamber samples was notable and relevant for public health consideration. C. jejuni
isolates had varying virulence patterns (n=15) and diverse pathogenic potential which are not always necessarily expressed in-vivo. The detected virulence genes and their patterns surely demonstrate the pathogenic potential of Campylobacter jejuni
isolated across the broiler chain.
CONFLICT OF INTEREST
The authors declare that they have no potential conflict of interest with respect to the authorship and/or publication of this article.
This research was supported by the Faculty of Veterinary Medicine – Skopje. The authors would like to express their gratitude to colleagues Prof. Kiril Krstevski and Prof. Igor Dzadzovski for their scientific advice and technical help.
LjA conceived and designed the study and wrote the manuscript. LjA, ZP, KB and MP performed the experiments. SM contributed to the final version. DJ gave critical revision. PS supervised the project and approved it for publishing.