# Molecular typing and virulence gene profiles of Campylobacter jejuni isolated from human, animals, and meat in northern Iran

G Divsalar, H Kaboosi, R Khoshbakht, H Shirzad-Aski, F Peyravii

Ghadikolaii

► To cite this version:

G Divsalar, H Kaboosi, R Khoshbakht, H Shirzad-Aski, F Peyravii Ghadikolaii. Molecular typing and virulence gene profiles of Campylobacter jejuni isolated from human, animals, and meat in northern Iran. Revue de Médecine Vétérinaire, 2019, 7 (170), pp.129-135. hal-04912071

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# Molecular typing and virulence gene profiles of *Campylobacter jejuni* isolated from human, animals, and meat in northern Iran

G. DIVSALAR<fnref n="1" />, H. KABOOSI<fnref n="1" />*, R. KHOSHBAKHT<fnref n="2" />, H. SHIRZAD-ASKI<fnref n="3" />, F. PEYRAVII GHADIKOLAI<fnref n="4" />

Corresponding author: h.kaboosi@iauamol.ac.ir

## SUMMARY

*Campylobacter jejuni* is one of the most important agents in gastrointestinal infections both in developed and developing countries. Determining the source of the human infections and the relationship of virulence gene properties and origin of the isolates are necessary epidemiologic data to control the infection in an area. We conducted a present study to genotype *C. jejuni* isolates recovered from human, broiler, cattle, poultry meat, and cattle meat in Mazandaran province, north of Iran. For this purpose, 100 isolates of *C. jejuni* (20 isolates from each source), which had been previously isolated, were subjected to Random Amplification of Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR), 16S rRNA Restriction Fragment Length Polymorphism (RFLP) analysis and six virulence genes (*cdtA*, *cdtB*, *cdtC*, *cadF*, *iamA* and *pldA*) pattern. In total, 98 isolates were typed using RAPD-PCR and 32 different genotypic patterns were observed. All 100 isolates showed similar enzymatic digestion pattern in 16S rRNA-RFLP technique by using *DdeI* and *AluI* enzymes. The *cadF* gene showed a 100% presence among the isolates. The prevalence of *cdtA*, *cdtB*, *cdtC*, *pldA*, and *iamA* genes was 97%, 97%, 96%, 72% and 60%, respectively. Ten different genotypic patterns (VP1-VP10) were identified from the presence of six virulence genes. According to the results, 16S rRNA-RFLP cannot type the isolates, but both RAPD and virulence typing methods showed significant relationships between some of the genotypes and a specific source. A potential role of poultry isolates in human infections was observed using these two methods. RAPD technique had the best results for typing of the *Campylobacter* isolates between these three methods.

**Keywords:** *Campylobacter jejuni*; RAPD-PCR; Virulence genes; RFLP; typing

## RÉSUMÉ

### Typage moléculaire et profils des gènes de virulence de *Campylobacter jejuni* isolés chez l'homme, les animaux et la viande dans le nord de l'Iran

*Campylobacter jejuni* est l'un des agents les plus importants dans les infections gastro-intestinales. La détermination de la source des infections humaines et la relation entre les propriétés des gènes de virulence et l'origine des isolats est une donnée épidémiologique nécessaire pour contrôler l'infection dans une région. Nous avons mené une étude sur le génotype des isolats de *C. jejuni* prélevés sur des humains, des poulets de chair, des bovins, de la viande de volaille et de la viande de bovins dans la province de Mazandaran, au nord de l'Iran. Cent isolats de *C. jejuni* (20 de chaque source précédemment isolés), ont été soumis à une amplification aléatoire de l'ADN (RAPD-PCR), à une analyse 16S du polymorphisme de l'ARNr (RFLP) et à six profils de virulence (*cdtA*, *cdtB*, *cdtC*, *cadF*, *iamA* et *pldA*). Au total, 98 isolats ont été typés à l'aide du RAPD-PCR et 32 profils génotypiques différents ont été observés. Les 100 isolats ont tous montré un schéma de digestion enzymatique similaire dans la technique 16S rRNA-RFLP en utilisant les enzymes *DdeI* et *AluI*. Le gène *cadF* a montré une présence de 100% parmi les isolats. La prévalence des gènes *cdtA*, *cdtB*, *cdtC*, *cdtC*, *pldA* et *iamA* était de 97%, 97%, 96%, 96%, 72% et 60%, respectivement. Dix modèles génotypiques différents (VP1-VP10) ont été identifiés à partir de la présence de six gènes de virulence. Selon les résultats, l'ARNr 16S-RFLP ne peut pas typer les isolats, mais les méthodes RAPD et le typage de virulence ont montré des relations significatives entre certains des génotypes et une source spécifique. En utilisant ces deux méthodes, un rôle potentiel des isolats de volaille dans les infections humaines a été observé. La technique RAPD-PCR a donné les meilleurs résultats pour le typage des isolats de *Campylobacter*.

**Mots-clés :** *Campylobacter jejuni* ; RAPD-PCR ; Gènes de virulence ; RFLP ; typage

## Introduction

*Campylobacter jejuni* is one of the major causes of acute gastrointestinal infections and some of the subsequent neuropathies such as Guillain–Barré syndrome in humans [5]. This bacterium is one of the most important foodborne pathogens around the world, which can be transmitted to humans through the digestive tract of several domestic animals and their related foods [11]. Although the cases of campylobacteriosis are sporadic and outbreaks are infrequent, the rate of the infections is increasing over the

last few years and has caused a lot of damages in terms of public health and the treatment of diseases [13, 21]. Most of *Campylobacter* spp. are present in the intestine of the animals without any symptoms [21]. Poultry, cattle, and pig are the most important reservoirs of the microorganism [9, 25]. Particularly, commercial farms of broilers and laying hens are the most important sources of *Campylobacter* for human [1]. To design an appropriate controlling and preventing system for *Campylobacter* infections, precise approaches for identifying, distinction, and typing of the strains are necessary for detecting disorders caused by campylobacters and discovering the source of the infections. Although

<footnote n="1">¹Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran.</footnote>

<footnote n="2">²Department of Pathobiology, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran.</footnote>

<footnote n="3">³Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran.</footnote>

<footnote n="4">⁴Department of Biology, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran.</footnote>
different typing methods have been used for campylobacters
during two recent decades [25, 26], it seems that choosing
an inexpensive and available genotyping method for all
laboratories around the world, which have the ability
to determine the source of the infections, is still a great
challenge. Random Amplification of Polymorphic DNA
Polymerase Chain Reaction (RAPD-PCR) technique is widely
used for typing of various bacteria, including pathogenic
Enterobacteriaceae and thermophilic Campylobacter spp. [3,
7]. In the present study, we aimed to determine the genotypic
characteristics and virulence properties of the C. jejuni
isolated from different origins in Mazandaran province,
north of Iran. For this purpose, genotyping of the C. jejuni
isolates from five various sources was done using RAPD-PCR
technique and the virulence genes patterns of the isolates in
related to the six virulence genes. In addition, Restriction
Fragment Length Polymorphism (RFLP) technique was
carried out using enzymatic digestions of a fragment of 16S
rRNA gene to obtain its possible genotyping potential in this
species.

**Materials and methods**

C. JEJUNI ISOLATES

Total of 100 C. jejuni isolates were considered in the present study. These isolates had been previously isolated and stored at -70 °C, in the microbiology laboratory of faculty of veterinary medicine, Mazandaran, Iran. The isolates were from five different sources, including poultry feces (n=20), cattle feces (n=20), poultry meat (n=20), cattle meat (n=20), and human diarrhea (n=20). All isolates had been obtained during of one-year sampling (2016-2017) in different places located in Mazandaran province, Iran. These places included slaughterhouses, supermarkets, and hospitals. All of the isolates had been previously recognized using specific genus and species primers (data still not published).

DNA EXTRACTION

Each isolate was subcultured in Brain Heart Infusion
(BHI) Broth (Himedia Labs, Mumbai, India) and was
incubated at 42 °C for 18 h. Each DNA was extracted using
the bacterial DNA extraction kit (CinnaPure DNA, SinaClon,
Iran), according to the manufacturer protocol.

RAPD-PCR

RAPD-PCR technique was carried out by a
random primer (M-13), previously described with the
5'-GAGGGTGGCGGTTCT -3' sequence [18]. The
thermocycler device program for this technique was as
follows: initial denaturation was first performed at 94 °C for
3 minutes, then 35 cycles were performed at a denaturation
temperature of 94 °C for 1 minute, 45 °C annealing
temperature for 45 seconds, and extension was carried out at
72 °C for 1 minute. After that, the final extension took place
at 72 °C and lasted 10 minutes. The PCR products obtained

by this method were analyzed using electrophoresis in 2.5%
agarose gel and the images were saved for further analysis.

16S RNA PCR-RFLP

For the digestion of a portion of the 16S rRNA gene
sequence to compare different patterns of the different
isolates, a relatively large area (856 bp) of this gene was initially
amplified by the primers of the Linton et al. with the forward
sequence of 5'-ATCTAATGGCTTAACCATTAAAC-3' and the reverse primer sequence of 5'-GGACGGTAACTAGTTTAGTATT-3' [19]. The
enzymatic digestion (RFLP) was then performed using
two restriction enzymes DdeI and AluI (Thermoscientific,
Fermentaz, Germany), according to the recommendations of
the manufacturer in separate reactions. For the DdeI enzyme
digestion, four units of the enzyme were mixed with 1.5 µl of
the enzyme buffer at a concentration of 10X and 5 µl of each
PCR reaction product and 9 µl of distilled deionized water.
The mixture was incubated at 37 °C for 15 hours. Stopping
of enzymatic activity immediately was done by placing
the samples at 65 °C for 20 minutes. For the AluI enzyme
digestion, 6 µl of each PCR reaction product was mixed with
1 µl of the enzyme specific buffer (10X) and five units (0.5 µl)
of the enzyme and the mixture was brought to a volume of 15
µl using 7 µl of distilled deionized water. Each mixture was
placed at 37 °C for 10 minutes. Each sample was incubated
at 65 °C for 20 minutes to stop the enzymatic activity. Each
product was separated using electrophoresis in 2.5% agarose
gel and the images were saved for further analysis.

VIRULENCE GENE PROFILES

Each isolate was examined for the presence of the six
virulence genes (cdtA, cdtB and cdtC, cadF, iamA, and pldA),
in order to determine the possible differences in the presence
of these genes in isolates of different origins. Simple PCRs
were performed using specific primers (Table I). Then, the
PCR product of the genes were evaluated and validated using
electrophoresis in 1.5% agarose gel with the aid of a marker
of 100 bp (Jena Bioscience, Germany). Different virulence
patterns were described according to the presence of the
genes.

GENOTYPING RESULTS ANALYSIS

The BioNumerics software version 6 (Applied Maths, Austin, TX, USA) was used to analyze the results of RAPD and RFLP images, as well as to determine and compare bands. The sensitivity of the software was adjusted and each gel and lane were manually curated as needed to confirm the stability across gels. The isolates were clustered according to the Dice coefficient by unweighted paired group method of arithmetic averages (UPGMA) and a 5% positional tolerance. The resulting dendrogram was partitioned at an 80% similarity cutoff to determine final groupings for genotyping methods and to recognize and found the relationship among isolates from various sources.
## STATISTICAL ANALYSIS AND EVALUATION OF GENOTYPING METHODS

The results of the study were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Chi-square (Independent Frequency Comparison Test) and Fisher's Exact (precision probability) tests were used to evaluate the relationship between different genotype patterns and the source of isolates. The *P* value of less than 0.05 was considered a significant difference. Finally, typability, reproducibility, and discriminative power of the genotyping methods were evaluated [10, 15].

## Results

Generally, 98% of the isolates were typed by RAPD-PCR technique and the same results were obtained when the technique was repeated again, while in this case, only 79% of the isolates showed the same previous patterns. In the software analysis, the isolates were placed in 32 clusters or groups (R1-R32). These results are shown in figure 1. The most prevalent RAPD cluster type was R4 (n=17), which

overlapped between *C. jejuni* belonged to cattle meat (n=7) and human (n=10) sources (figure 1). Patterns R9 and R26 were only observed in cattle and human, respectively. R11 and R24 RAPD patterns were observed only in cattle meat isolates. R3 and R7 patterns only belong to the poultry isolates and R10, R14, and R18 patterns belong to the poultry meat origin (figure 1). Contrary to this technique, all isolates had a similar pattern using 16S rRNA PCR-RFLP genotyping method. Therefore, using two *DdeI* and *AluI* enzymes digestion, no difference was observed between the isolates.

The results of the presence of the six different virulence genes in *C. jejuni* isolates from various sources are shown in Tables II and III. The *cadF* gene showed 100% presence among the isolates. The prevalence of *cdtA*, *cdtB*, *cdtC*, *pldA*, and *iamA* genes were 97%, 97%, 96%, 72%, and 60%, respectively. Ten different genotypic patterns/profiles (VP1-VP10) were identified by using the presence of the six genes among the isolates (figure 2). The most prevalent virulence pattern was VP1 (n=47), which was present among all five sources with the frequency of 14, 14, 12, four, and three for poultry, human, poultry meat, cattle meat, and cattle, respectively. VP7, VP9 and VP10 patterns were existence only among

<table><caption>TABLE I: Primer sequences used to detect *C. jejuni* virulence genes</caption><thead><tr><th>Primer name</th><th>Sequence (5' to 3')</th><th>Target gene</th><th>Annealing temperature</th><th>Product size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>DS-18</td><td>CCTTGTGATGCAAGCAATC</td><td>cdtA</td><td>49 °C</td><td>370</td><td>[14]</td></tr><tr><td>DS-15</td><td>ACACTCCATTTGCTTTCTG</td><td>cdtA</td><td>49 °C</td><td>370</td><td>[14]</td></tr><tr><td>cdtB-113</td><td>CAGAAAGCAAATGGAGTGTT</td><td>cdtB</td><td>51 °C</td><td>620</td><td>[9]</td></tr><tr><td>cdtB-713</td><td>AGCTAAAAGCGGTGGAGTAT</td><td>cdtB</td><td>51 °C</td><td>620</td><td>[9]</td></tr><tr><td>cdtC-192</td><td>CGATGAGTTAAAACAAAAAGATA</td><td>cdtC</td><td>48 °C</td><td>182</td><td>[9]</td></tr><tr><td>cdtC-351</td><td>TTGGCATTATAGAAAATACAGTT</td><td>cdtC</td><td>48 °C</td><td>182</td><td>[9]</td></tr><tr><td>iamA F</td><td>GCGCAAAATATTATCACCC</td><td>iamA</td><td>52 °C</td><td>518</td><td>[6]</td></tr><tr><td>iamA R</td><td>TTCACGACTACTATGCGG</td><td>iamA</td><td>52 °C</td><td>518</td><td>[6]</td></tr><tr><td>pldA-84</td><td>AAGCTTATGCGTTTTT</td><td>pldA</td><td>45 °C</td><td>913</td><td>[9]</td></tr><tr><td>pld-981</td><td>TATAAGGCTTTCTTCA</td><td>pldA</td><td>45 °C</td><td>913</td><td>[9]</td></tr><tr><td>cadF F</td><td>TTGAAGGTAATTTAGATAT</td><td>cadF</td><td>42 °C</td><td>400</td><td>[16]</td></tr><tr><td>cadF R</td><td>CTAATACCTAAAGTTGAAAC</td><td>cadF</td><td>42 °C</td><td>400</td><td>[16]</td></tr></tbody></table>

<table><caption>TABLE II: Occurrence of six virulence genes among *C. jejuni* isolates from various sources.</caption><thead><tr><th rowspan="2">Source</th><th colspan="6">Occurrence of virulence genes (%)</th></tr><tr><th>cdtA</th><th>cdtB</th><th>cdtC</th><th>cadF</th><th>iamA</th><th>pldA</th></tr></thead><tbody><tr><td>Poultry (N = 20)</td><td>20 (100)</td><td>20 (100)</td><td>20 (100)</td><td>20 (100)</td><td>15 (75)</td><td>18 (90)</td></tr><tr><td>Poultry meat (N = 20)</td><td>20 (100)</td><td>20 (100)</td><td>20 (100)</td><td>20 (100)</td><td>12 (60)</td><td>19 (95)</td></tr><tr><td>Cattle (N = 20)</td><td>18 (90)</td><td>18 (90)</td><td>20 (100)</td><td>20 (100)</td><td>6 (30)</td><td>7 (35)</td></tr><tr><td>Cattle meat (N = 20)</td><td>19 (95)</td><td>20 (100)</td><td>16 (80)</td><td>20 (100)</td><td>10 (50)</td><td>11 (55)</td></tr><tr><td>Human diarrhea (N = 20)</td><td>20 (100)</td><td>19 (95)</td><td>20 (100)</td><td>20 (100)</td><td>17 (85)</td><td>17 (85)</td></tr><tr><td><strong>Total (N = 100)</strong></td><td><strong>97</strong></td><td>97</td><td>96</td><td>100</td><td>60</td><td>72</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>FIGURE 1: RAPD dendrogram of *C. jejuni* isolates, collected from poultry, poultry meat, cattle, cattle meat, and human, using the UPGMA analysis. P: poultry; PM: poultry meat, C: cattle; CM: cattle meat; H: human</figcaption></figure>

cattle meat source. VP8 pattern was present in human samples. The cattle isolates with eight different patterns were the most diverse isolates and the poultry meat isolates with three patterns showed the lowest diversity in the virulence gene profile (Table III). According to the statistical analysis, human isolates had a significant correlation with the VP1 virulence typing pattern. In addition, Poultry and poultry meat isolates had a significant correlation with this VP1 virulence pattern as well as RAPD-

1cluster ($P \le 0.05$). Isolates obtained from poultry meat had a significant correlation with the presence of *pldA* virulence gene ($P \le 0.05$). The cattle isolates had a significant correlation with the VP3 pattern of virulence genes ($P \le 0.05$). Cattle meat isolates showed a significant correlation with the pattern of VP2 ($P \le 0.05$). Moreover, human isolates showed a significant correlation with the presence of *iamA* gene ($P \le 0.05$).
<figure><img src="image_3.png" /><figcaption>FIGURE 2: The diagram of the relationship between different sources and virulence profile typing.</figcaption></figure>

RAPD technique showed 0.921 discriminative power with the 98% typeability, while the 16S rRNA RFLP did not show acceptable results in discrimination and typeability aspects. The summarized results of typeability, reproducibility, and discriminatory power of the genotyping methods are shown in Table IV.

Discussion

Typability of a genotypic method is the first important factor in such techniques. In this regard, the RAPD method using M-13 random primer showed high and relatively suitable discriminatory power and had a good typeability. Findik et al. reported the same results. They were analyzed 168 isolates of C. jejuni using the same primer and reported 19, 18, 17, 18, and 6 genotypes in humans, cattle, sheep, dogs, and poultry, respectively. Moreover, similar to the present results, their study revealed a high genetic heterogeneity among the isolates with different sources. For example, the dominant genotype in human isolates was found only in 4 isolates [12]. In a study in 2006, Acik and Cetinkaya also typed 115 and 103 isolates of cattle and sheep and found 37 and 21 different patterns, respectively. Using RAPD typing method, they described a vast genetic heterogeneity, which was also observed in this study [2].

It seems that there are many variables that can affect the patterns of genotyping methods such as RAPD method, for instance, geographical differences, sample origins, sample differences, type of primers, and ultimately PCR conditions. The most important feature of the RAPD method is its low cost and high-speed performance; however, this method hasn't high reproducibility power [4]. Therefore, different patterns can be seen in the re-implementation of the method on one

<table><thead><tr><th rowspan="2">Patterns of the virulence genes</th><th rowspan="2">Number in the group</th><th rowspan="2">Name</th><th colspan="5">Number of sample/Source of the sample</th></tr><tr><th>Poultry</th><th>Poultry meat</th><th>Cattle</th><th>Cattle meat</th><th>Human</th></tr></thead><tbody><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/cdtC<sup>+</sup>/cadF<sup>+</sup>/iamA<sup>+</sup>/pldA<sup>+</sup></td><td>47</td><td>VP1</td><td>14</td><td>12</td><td>3</td><td>4</td><td>14</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/cdtC<sup>+</sup>/cadF<sup>+</sup>/ - /pldA<sup>+</sup></td><td>22</td><td>VP2</td><td>4</td><td>7</td><td>4</td><td>5</td><td>2</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/cdtC<sup>+</sup>/cadF<sup>+</sup>/ - / -</td><td>11</td><td>VP3</td><td>1</td><td>1</td><td>6</td><td>3</td><td>-</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/cdtC<sup>+</sup>/cadF<sup>+</sup>/iamA<sup>+</sup> - / -</td><td>10</td><td>VP4</td><td>1</td><td>-</td><td>3</td><td>3</td><td>3</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/cdtC<sup>+</sup>/cadF<sup>+</sup>/ - / -</td><td>3</td><td>VP5</td><td>-</td><td>-</td><td>2</td><td>1</td><td>-</td></tr><tr><td>cdtA<sup>+</sup>/ - /cdtC<sup>+</sup>/cadF<sup>+</sup>/ - / -</td><td>2</td><td>VP6</td><td>-</td><td>-</td><td>2</td><td>-</td><td>-</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/ - /cadF<sup>+</sup>/iamA<sup>+</sup> - / -</td><td>2</td><td>VP7</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td></tr><tr><td>cdtA<sup>+</sup>/ - /cdtC<sup>+</sup>/cadF<sup>+</sup>/ - /pldA<sup>+</sup></td><td>1</td><td>VP8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/ - /cadF<sup>+</sup>/iamA<sup>+</sup>/pldA<sup>+</sup></td><td>1</td><td>VP9</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td></tr><tr><td>cdtA<sup>+</sup>/cdtB<sup>+</sup>/ - /cadF<sup>+</sup>/ - /pldA<sup>+</sup></td><td>1</td><td>VP10</td><td>-</td><td>-</td><td>-</td><td>1</td><td>-</td></tr></tbody><tfoot><tr><td>Total</td><td>100</td><td>10</td><td>4</td><td>3</td><td>6</td><td>8</td><td>4</td></tr></tfoot></table>

VP: virulence pattern; P: poultry; PM: poultry meat, C: cattle; CM: cattle meat; H: human.

<table><caption>TABLE III: virulence patterns of the C. jejuni isolated from five sources</caption><thead><tr><th>Typing method</th><th>Discriminative power (0-1)</th><th>Reproducibility (%)</th><th>Typability (%)</th><th>Cost level</th></tr></thead><tbody><tr><td>RAPD-PCR</td><td>0.9216</td><td>79</td><td>98</td><td>low</td></tr><tr><td>Virulence-typing</td><td>0.7137</td><td>100</td><td>100</td><td>medium</td></tr><tr><td>16S rRNA PCR-RFLP</td><td>0</td><td>100 (unacceptable)</td><td>100 (unacceptable)</td><td>medium</td></tr></tbody></table>

<caption>TABLE IV: characteristics of three genotyping methods of C. jejuni.</caption>
sample in an outbreak. Such results may lead to inappropriate interpretations and often restrict the utility of the method to a region or a specific laboratory. Therefore, besides a good had a goo typeability power of the method, the value of this technique can decline within the scope of internal and regional examination of a group of isolates, especially when the isolates obtained from an outbreak or sudden occurrence. In the present study, the reproducibility of the RAPD method similar to the studies was not 100% [2, 12].

The results of FRLP analysis using two *DdeI* and *AluI* restriction enzymes did not show any difference between the isolates with different origins. It seems that this typing method particularly with these enzymes, may not be suitable for the detection and differentiation of strains of one species, and as described previously, can be more useful in differentiating between two or more species or genus [22, 27].

In the statistical analysis of the results, appreciated observations were made of the presence of these genotypes in different isolates. In fact, the main difference between the total of isolates was not the presence of cytolethal distending toxin (CDT) and *cadF* genes. It seems that the significant differences of the strains with various origin were about invasion associated marker (*iamA*) and phospholipase producing gene (*pldA*), which both of these genes are important invasion and pathogenicity factors in particularly human [6, 9]. Some of the virulence genotypes (profiles) were dedicated to a specific source. For example, VP7, VP9, and VP10 genotypes were found only in beef isolates, and the VP8 genotype was specific to a human isolate. Also, based on the results, there were significant relationships between some of the genotypes and a specific source. The genotype of VP1 was found as a dominant genotype among the isolates of poultry, poultry meat, and humans. This connection is alarming and it strengthens the possibility that isolates from poultry can be a source of human infection in the area. Moreover, the presence of appropriate virulence factors of the microorganism in these sources also increases their pathogenicity in humans [17].

Mullner et al. investigated the source of campylobacteriosis in New Zealand using the Multi Locus Sequence Typing (MLST) method and their data analysis showed the broiler poultry as the most important source of human infection in that area [23]. In 2008, Wilson et al. stated that poultry, cattle, and sheep are the origin of 97% of human campylobacteriosis in England, and only 3% of infection cases were associated with environmental sources such as contaminated water. They also described the food cycle as the most important route and pathway of transmission of *Campylobacter* infection. They also stated that it is necessary to prevent the transmission of the disease from livestock to humans and follow-up ways to process and produce food, in order to fight infection [28]. Both results were in accordance with the present result. Another study by Shepard et al. was evaluated the source of human infection, using genotyping of 5674 different *Campylobacter* isolates by means of the MLST method. They indicated that poultry and

ruminants are the most important sources of transmission of this infection [25].

In this study, both RAPD and virulence typing methods highlighted the effective role of poultry in human infection. Both these methods, unlike the other method (RFLP), had more acceptable results and can be used to type *C. jejuni* at the strain level.

For comparison of different types of genotyping methods to establish source tracing system, it should be possible to examine each method in a number of different aspects, such as: the ease of carrying out the technique, the ability to transfer (meaning data transfer to the computer and databases for analysis), proper access (meaning Simplification in laboratories), adaptability (meaning proper level of separation and strain discrimination), and sustainability (no change in recombination and entry of new DNA) [26]. In the present study, RAPD technique displayed appropriate results in comparison with two others.

## Conclusion

Based on the results, the RAPD typing method showed an appropriate results for discriminatory and typeability. According to the evaluation of virulence properties and RAPD analysis of the isolates, some significant similarities were observed between human and poultry *C. jejuni* isolates. In addition, in this study, the typing method using 16S rRNA enzymatic digestion did not show respectable results and it was found that this method would not apply to type strains of one species (*C. jejuni*) from various sources.

## Acknowledgment

This study was supported by Islamic Azad University of Ayatollah Amoli branch and Amol University of Special Modern Technologies.

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