Prevalence, Virulence, and Antimicrobial Resistance of Campylobacter Species Isolated From Carcasses of Camels Slaughtered in Slaughterhouses of Chaharmahal and Bakhtiari Province, 2018-2019

Background and aims: Gastritis is basically caused by Campylobacter coli and jejuni, and usually occures after the consumption of raw animal products. Methods: This study investigated the prevalence, virulence, and antimicrobial resistance of Campylobacter species isolated from slaughtered animals in Juneqan, Farrokhshahr, Saman, and Lordegan slaughterhouses in Chaharmahal and Bakhtiari Province of Iran. From 40 camels, 5 samples of liver, neck meat, kidney, heart, and rectal contents were taken from each carcass. The obtained samples were cultured and then the PCR was performed for them and, finally, the toxin genes of virulence and resistance against antibiotics were examined. Results: Out of 19 Campylobacter specimens isolated, 8 specimens were coli and 11 ones were jejuni. It was also found that the infection with Campylobacter in the carcasses was the highest in warmer


Introduction
Foodborne illnesses are important public health problems that cause considerable economic damages. 1,2 Campylobacter is a rod-shaped, Gram-negative, and non-spore-forming bacterium belonging to the Enterobacteriaceae family. It has various hosts, and constitutes a common zoonotic pathogen. 3,4 The two important species C. jejuni and C. coli are the causal agents of most Campylobacter infections that are transmitted to humans by animal vectors. 5,6 Campylobacter is responsible for 2%-35% of bacterial diarrhea in human. [7][8][9][10][11] The infective dose of Campylobacter is very low. 10,11 The bacterium affects livestock and results in economic loss, and is the causative agent of food poisoning in humans. 12,13 These infections are clinically manifested as gastroenteritis, typhoid fever, and septicemia with local lesions. Arbitrarily use of antibiotics in the livestock and poultry industry as well as in human communities have become troublesome, leading to the emergence of Campylobacter resistance to various antibiotics. 8 Consumption of raw and undercooked meat is the main transmission route of Campylobacter to humans, but unpasteurized milk and raw vegetables can also be a source of these bacteria and cause diseases in humans. Campylobacter infections often occur during traveling, which they are also referred to as traveler's diarrhea.
The rate of Campylobacter infections is 380 per 100 000 population. 14 Campylobacteriosis is the dominant bacterial infection in food materials and is considered the major public health problem in Europe and many other countries worldwide. 12 Campylobacter has been isolated from intestinal contents, liver, gallbladder, and feces of livestock. Also, in a few cases, it has been isolated from carcasses in slaughterhouse cold rooms. 1 In a study conducted in Egypt, Campylobacter was found in 20% of the fecal and up to 33% of the meat samples. 14 The infection with Staphylococcus aureus and Campylobacter spp in camels is of the highest importance. 15,16 This study aimed to determine the prevalence, intensity, and antimicrobial resistance of Campylobacter isolated from carcasses of slaughtered camels in Chaharmahal and Bakhtiari province, Iran.

Materials and Methods
Our study samples were obtained from the liver, neck meat, kidney, heart, and rectal contents of the carcasses of 40 camels slaughtered in Juneqan, Farrokhshahr, Saman, and Lordegan slaughterhouses from September 2018 to September 2019. The samples were cultured and polymerase chain reaction (PCR) was performed. As for the isolation of campylobacter species: first, all samples were homogenized, and in each one, 10 g was added to 90 mL Campylobacter Enriched Broth (Preston enrichment broth base, Himedia, Mumbai, India, M899); then the selected Campylobacter supplement (Himedia, Mombia, India, FD042) and 25mL defibrinated sheep blood were added to each 475 mL of the medium. After 24 hours of incubation, 0.1 mL of it added to the selective Campylobacter media (Himedia, Mumbai, India, M994) was enriched with antibiotic supplements (Himedia, Mumbai, India, FD006) and 5% sheep's defibrinated blood, and incubated for 48 hours at 42°C. Single-growing colonies were studied to confirm and separate Campylobacter species in terms of warm staining, catalase production, oxidase, hydrolysis of hippurate, and resistance to cephalothin.

DNA Extraction and PCR Test
DNA of the confirmed colonies was extracted using the DNA extraction kit (Cinna Gen, Iran). The PCR was performed as described by Denis et al 17 . To conduct the PCR reaction, the final reaction volume was considered 25 μL, including 20 ng of template DNA, 2 mM MgCl2, 25 pmol of each primer, one Taq polymerase enzyme unit, and 200 μM dNTP mixture. Table 1 shows the size of the PCR product for each sample. To confirm the presence of amplified fragment, 20 µL of the PCR product was electrophoresed on 1.5% agarose gel containing ethidium bromide in the presence of 100 bp DNA marker at a constant voltage of 80 V. Other primers are listed in Table  2. The main method of this study was PCR Test.
Antimicrobial susceptibility test was performed using the disk diffusion method on Muller Hinton medium (HiMedia, Laboratories, Mumbai, India) enriched with 5% sheep defibrinated blood, according to the method proposed by CLSI (Clinical and Laboratory Standards Institute, 2006). The antibiotic discs used in this study were manufactured by Indian HiMedia companies (HiMedia, Laboratories, Mumbai, India). The type and concentration of each antibiotic used were as follow: Nalidixic acid (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), tetracycline (15 µg), streptomycin (30 µg), ampicillin (10 µg), amoxicillin (30 µg), gentamicin (10 µg), and chloramphenicol (30 µg). After culturing and disking at 42°C under microaerophilic conditions for 48 hours, the plates were incubated. After incubation, non-growth areas around antibiotic discs were measured by a KT model caliper made in China. The sensitivity of Campylobacter strains to each antibiotic was then compared to the pattern presented by CLSI. The PCR method was used to trace the virulence genes of Campylobacter isolated from the studied samples based on the study conducted by Bang et al. 5 The samples were taken from neck muscles in the slaughterhouse after being washed. Campylobacter species as the pathogen-dependent variable were isolated by the culture method and biochemical tests, confirmed by these methods, and examined in terms of presence of virulence genes. Antimicrobial resistance of Campylobacter species was evaluated by the disk diffusion method.
As for the livestock slaughtered in Chaharmahal and Bakhtiari province's slaughterhouses, sampling was conducted from neck, heart, liver, kidney, and intestine contents. Statistical analyses were conducted using SPSS software 16.0 (SPSS Inc, Chicago, IL.), and chi-square test as well as Fisher's exact two-tailed test analysis were performed.

Results
Out of 40 meat samples, 5 samples were infected with Campylobacter (1 with C. coli and 4 with C. jejuni); out of 40 liver samples, 6 ones were Campylobacter positive (4 with C. coli and 2 with C. jejuni); as for 80 kidney and heart samples, no Campylobacter (C. jejuni or C. coli) was isolated; and out of 40 rectal content samples, 8 ones had Campylobacter (3 with C. coli and 5 ones with C. jejuni).  Overall, 19 samples out of the 200 ones were found positive for Campylobacter (8 with C. coli, and 11 with C. jejuni). In other words, 4% of the total infected samples contained C. coli and 5.5% of them contained C. jejuni (see Table 3 and Figure 1). Out of the 5 Campylobacter-infected camel meat samples, C. coli was found in 20% of the samples and C. jejuni was observed in 80% of them. Out of 6 Campylobacter-infected camel liver samples, C. coli was found in 66.6% of them and C. jejuni was detected in 33.3% of them. Neither C. coli nor C. jejuni was found in the samples from camels' kidney and heart (0%). As for the Campylobacter-infected camel rectal content samples, C. coli and C. jejuni were found in 37.5% and 62.5% of them, respectively. Out of the total 19 infected camel samples, C. coli was found in 42.1% of the samples and C. jejuni was observed in 57.8% of them (P=0.04; Table 4).
Presences of the genes effective in motility (flaA), adhesion (cadF), and cytotoxin production (cdtB, cdtA) were confirmed. The ability of Campylobacter strains in producing toxins is also important in the infection process.
In Campylobacter infections, the cytolethal distending toxin consisting of the subunits CdtA and CdtB is the most important characteristic showing the presence of    Campylobacter toxins (Figure 2). Campylobacter isolated from liver samples indicated that cadF and FlaA were the most frequent genes followed by cdtA gene (83.3%). The cdtA gene was found in 75% of the rectal content samples. These genes were least frequently detected in kidney and heart samples containing Campylobacter (P= 0.03; Table 5).
As shown in Tables 5 and 6, the frequencies for the cdaF and flaA genes in the camels were both 100% (Figure 3), whereas those for the cdtA and cdtB genes were 73.6%, 47.3%, respectively (P=0.03; Table 5). The most isolated genes of Campylobacter detected from rectal contacts was 100%, and the least isolated genes of Campylobacter detected from both kidneys and hearts were both 0% ( Table 6).
The rates of antimicrobial resistance in C. jejuni were 54.54% for erythromycin, 18.18% for meropenem, 9.09% for imipenem and nalidixic acid, 45.45% for amoxicillin and streptomycin, 81.81% for ciprofloxacin, 27.27% for norfloxacin and amikacin, 36.36% for gentamicin, 90.9% for tetracycline, 81.8% for chloramphenicol, and 54.54% for ampicillin. The highest antibiotic resistance rate of C. jejuni was recorded for tetracycline (90.9%), and the lowest one was recorded for nalidixic acid and imipenem (9.09%).
The rate of antimicrobial resistance in C. coli isolated from the samples of camels slaughtered in Chaharmahal and Bakhtiari province was 25% for erythromycin and norfloxacin, 12.5% for meropenem, amikacin, and nalidixic acid, 0% for imipenem, 50% for amoxicillin, gentamicin, and streptomycin, 62.5% for ciprofloxacin, 87.5% for tetracycline, and 75% for ampicillin. The highest resistance rate was observed for tetracycline (87.5%) and the lowest one was found for imipenem (0%).
Several studies have already investigated the contamination of camel carcasses with this pathogen in slaughterhouses in different seasons; therefore, our study results only covered the contamination in different seasons as follows: Spring: The highest and lowest percentages of infection with Campylobacter were observed at the slaughterhouse in Lordegan (14.4%) and Juneqan (4.8%), respectively, which were significantly different. In this season, the largest and smallest percentages of infection with C. coli were found in Saman (62.5%) and in Juneqan (33.3%),    respectively, which were also significantly different. As for C. jejuni, the highest and lowest percentages of infection were recorded in Juneqan (66.6%) and Saman (37.5%), respectively, which were significantly different. Summer: The largest and smallest percentages of infection with Campylobacter were observed in Lordegan (18.4%), and Saman & Juneqan (0.8%), respectively, which were significantly different. The highest and lowest percentages of infection with C. coli were detected in Lordegan (56.5%) and Saman (20%), respectively. As for C. jejuni, the largest and smallest percentages of infection were recorded in Saman (80%) and Lordegan (43.4%), respectively, which were significantly different.
Autumn: The smallest and largest percentages of Campylobacter infection were discovered in Juneqan (0%) and Lordegan (88%), respectively, which were significantly different. The highest and lowest percentages of infection with C. coli were found in Farrokhshahr (57.1%) and Juneqan, which were significantly different. The lowest and highest percentages of infection with C. jejuni were recorded in Juneqan (0%) and (54.5%) Lordegan, respectively.
Winter: The smallest and the largest percentages of Campylobacter infection were recorded in Juneqan and Lordegan, respectively, which were significantly. The lowest percentages of C. coli and C. jejuni infections (0%) were those for Juneqan, and the highest percentages of C. coli and C. jejuni infections (75% and 50%, respectively) were those for Farrokhshahr and Saman, respectively, which were significantly different.

Discussion
According to Table 4 and out of 5 Campylobacter-infected camel meat samples, C. coli was found in 20% of the samples and C. jejuni was detected in 80% of them. Out of 6 Campylobacter-infected camel liver samples, C. coli was found in 66.6% of the samples and C. jejuni was detected in 33.3% of them. Neither C. coli nor C. jejuni was found in the camels' kidney and heart samples (0%). In the Campylobacter-infected camel rectal content samples, C. coli was discovered in 37.5% of the samples and C. jejuni was observed in 62.5% of them. Out of 19 infected camel samples, C. coli was found in 42.1% of the samples and C. jejuni was detected in 57.8% of them (P=0.04).
A study in Egypt indicated that 20% of the fecal samples were infected with Campylobacter, whereas   up to 33% of camel meat samples were infected with Campylobacter; and 15% of the liver samples were infected with Campylobacter. Moreover, C. jejuni was detected in 26.3% of the infected meat samples, and C. coli and C. lari were observed in 57.9% and 15.7% of them, respectively. 14 In the present study, 42% and 57% of the Campylobacterpositive samples were infected with C. coli and C. jejuni, respectively. Out of 19 Campylobacter-infected samples, 5 were from meat (one sample with C. coli and 4 samples with C. jejuni). In other words, 20% and 80% of meat samples were infected with C. coli and C. jejuni, respectively, indicating a significant difference between the two Campylobacter spp. and concerning their presence in Campylobacter-infected camel meat (P=0.03). C. coli was detected in a higher percentage of the infected meat samples in this study compared to the study conducted in Egypt, demonstrating a difference between Iran and Egypt regarding the prevalence of Campylobacter spp.

Distribution of the Virulence Genes in the Campylobacter Isolates
In a study by Casabonne et al in 2016, cdtA, flaA, and cadF genes were found in 100% of the isolates (the survey was done in Poland). 7 Bang et al reported that cdtA genes was detected in 90% of Campylobacter isolates (the survey was done in Denmark). 5 In the present study, these genes were detected in 19 Campylobacter-infected samples. All 19 samples contained the cadF and flaA genes, whereas the frequencies of cdtA and cdtB genes were 79.3% and 61.3%, respectively. Therefore, there were no significant differences between the cadF and flaA genes concerning their presences in the infected samples. However, there was a significant difference between cdtA and cdtB genes in terms of their frequencies (P=0.03).  16 However, the resistance rate to this antibiotic was 15.78% (P:0.04) in our study.

Antimicrobial Resistance Among Campylobacters isolated From the Infected Samples
Imipenem: In Finland, Lehtopolku et al 16 showed that Campylobacter was completely susceptible to imipenem under in vitro conditions, whereas in the present study, it was 5.26%.
Amoxicillin: The study on samples from camels in Iran by Rahimi et al 8 showed that resistance to this antibiotic was 6.5%. However, in this study it was 47.36% (45.45% in C. jejuni and 50% in C. coli), which suggested a significant difference between the two reports.
Norfloxacin: Rahimi et al 8 demonstrated that the resistance rate to norfloxacin in Campylobacter-positive samples was 32.7%. It was 26.31% in the present study, which indicated the increased resistance of Campylobacter to this antibiotic.
Amikacin: In the study by Gwida et al, 15 Campylobacter was 100% resistant to amikacin. In the present study, however, the resistance was 21.05%, which indicated the higher resistance of microorganisms to amikacin in Egypt compared to Iran.
Gentamicin: Rahimi et al 8 showed that the resistance rate to this antibiotic in Campylobacter-positive camel samples was 3.2%, while it was 42.01% in the present study. These findings showed a considerable increase in the resistance of Campylobacter to this antibiotic.
Tetracycline: The study by Rahimi et al 8 determined that the resistance rate to this antibiotic was 75%. In our study, however, it was 89.47% indicating the increased resistance of Campylobacter to this antibiotic in various geographic regions.
Nalidixic acid: Gwida et al showed that the resistance rate to this antibiotic in Campylobacter-positive samples was 75%, 15 while it was 10.52% in our study.
Chloramphenicol: Rahimi et al 8 in Iran revealed that the resistance rate to this antibiotic in Campylobacter-positive samples was 6.5%. In the present study, however, it was higher (84.21%) indicating a higher resistance of this microorganism to this antibiotic.
Ampicillin: Gwida et al 15 reported that the resistance rate to this antibiotic in Campylobacter-positive camel samples was 90%. In our study, however, it was 63.15% suggesting a higher resistance of this microorganism to this antibiotic in Egypt compared to Iran.
Streptomycin: Gwida et al 15 reported that the resistance rate to Amikacin in Campylobacter-positive samples was 100%, whereas the resistance rates to streptomycin in these samples were 0.9% and 0% in C. jejuni and C. coli, respectively. 11 In the present study, the resistance rate to streptomycin in Campylobacter-positive samples was 47.36%, which suggested that this microorganism was more resistant to these antibiotic studies in Egypt compared to Iran.
Ciprofloxacin: Lehtopolku et al 16 reported that the resistance rate of Campylobacter isolates to this antibiotic was 73.68%, which indicated a higher resistance.

Conclusion
Our study results highlighted the necessity of performing further researches on how to combat the summer peak of Campylobacter in camels to improve the safety of the human food supply. Campylobacter is difficult to isolate, grow, and identify. Public health reference laboratories can play a key role in standardizing, validating and disseminating methods for clinical diagnosis, as well as in supporting periodic or sentinel targeted surveillance studies. This study showed the importance of the camel meat products as potential sources of Campylobacter spp. infection in people who had consumed the given products. It was recommended that coordinated measures be urgently adopted in order for reducing or eliminating the risks posed by this organism at a number of stages in the food chain. These included good agricultural practice, sound manufacturing practice, and hazard analysis in slaughters.