Abstract: Background and Objective: Campylobacter jejuni (C. jejuni) is a commensal microorganism in birds that causes diarrhea due to intestinal inflammation, which leads to feet damage from standing on wet litter. This study aimed to investigate the prevalence and virulence of C. jejuni in broiler chickens, as well as determine histopathological changes in the chicken gut following infection. Materials and Methods: A total of 200 infected broiler chicken samples (100 cecal contents, 50 livers and 50 spleens) were collected from different farms at Dakahlia Governorate in Egypt. Broilers samples were subjected to Campylobacter isolation procedures. Campylobacter jejuni isolates were distinguished from other Campylobacter spp. using the uniplex PCR-targeting mapA gene. Campylobacter jejuni isolates were also evaluated for the presence of cdtA and flaA genes. Evaluation of gut histopathological changes after C. jejuni infection was carried out in twenty day-old broiler chicks. Results: In total, sixty-eight C. jejuni isolates (18 isolates from the liver samples, 12 from spleen samples and 38 from cecal contents) were recovered with an overall prevalence rate of 34%. The flaA gene was successfully identified in all Campylobacter isolates, while cdtA, was identified in only 62 (91.17%) isolates. Several pathological changes and inflammatory responses were found in the chicken gut as a result of C. jejuni infection. Conclusion: The prevalence of flaA and cdtA revealed a high rate of adherence and cytotoxicity-associated genes. In addition, several histopathological changes were found in the chicken gut. These findings confirmed that C. jejuni infection has a significant impacton poultry health and welfare and C. jejuni is a harmful gut commensal microorganism.
INTRODUCTION
Campylobacter is one of the most important bacterial sources of human enteritis worldwide and it has been isolated from a variety of animal species, including, poultry, cattle, pigs, sheep, pets, wild birds and rodents1-3. Chicken is considered the main source of Campylobacter and may responsible for approximately 70.9% of human cases, as contaminated chicken meat and meat by-products often cause human infections4,5. It is difficult to control the spread of Campylobacter in the slaughterhouse and in kitchens; however, controlling Campylobacter on the farm may help reduce the number of bacteria in food processing plants6,7.
The mechanisms of disease of C. jejuni starts with colonization in the chicken gut and adhesion and invasion of the gut epithelial cells followed by cytotoxin production8. Once colonization is established, Campylobacter can multiply rapidly in the cecal contents9. While C. jejuni colonizes chicken ceca and small intestines, it may also be isolated from different places in the chicken gut, as well as from internal organs such as spleen and liver10. Campylobacter may present in poultry internal organs tissues via bacterial translocation by which it can cross the intestinal barrier of humans and animals and invade internal organs; the lymphatic path is thought to be the primary route of translocation11.
Detection of virulence in C. jejuni can be used to evaluate the potential risk of poultry as a source of Campylobacter infection12. Motility is one of the main factors of Campylobacter pathogenicity and it can be identified by its spiral form and flagella bundles on cell tips. Due to these features, Campylobacter can move against peristaltic movements and colonize intestinal cells. The flagellum is built from a protein known as flagellum which is encoded by the flaA and flaB genes. The flaA gene is expressed at higher rates than flab and thus is essential to Campylobacter motility13. Most C. jejuni strains have relatively higher cytolethal distending toxin (CDT) activity comparing to C. coli strains; the C. jejuni CDT is encoded by a three-gene operon (cdtABC)14-16.
The aims of this study were to characterize C. jejuni isolated from chicken cecal contents, determine whether Campylobacter can be isolated from the liver and spleen and determine the prevalence of virulence genes. In addition, we examined intestinal epithelial changes caused by C. jejuni infection.
MATERIALS AND METHODS
Sample collection: A total of 200 infected broiler chicken samples (100 cecal contents, 50 livers and 50 spleens) were collected from different broiler farms at Dakahlia Governorate, Egypt from October 2016 to February 2017. Cecal contents and internal organs from each chicken were individually packed into sterile plastic bags. All samples were immediately transported to the laboratory in an ice box to isolate C. jejuni.
Isolation and identification of C. jejuni isolates: Campylobacter isolation procedures were performed according to ISO 10272-1:200617. Samples from cloaca swabs were dissolved into Bolton broth (CM0983, Oxoid) with Campylobacter growth supplement (SR0183, Oxoid) and incubated at 42°C for 24 h under microaerobic conditions followed by streaking into mCCDA agar plates (CM0739; Oxoid/UK) with a selective supplement (SR0155, Oxoid) according to the manufacturer’s instructions. For internal organs (liver and spleen), 25 g of each sample were homogenized for 1min in stomacher (Stomacher 400 Lab Blender) with Bolton broth; then, the homogenate was transferred to screw-capped sterile bottle. The bottles were then incubated aerobically at 37°C for 4 h followed by 42°C for 48 h. Approximately 0.1-0.2 mL of each samples’ enrichment broth was streaked into mCCDA agar plates and then incubated at 42°C for 48 h under microaerobic conditions using Oxoid Campy Gen (CN035A, Oxoid). Presumptive Campylobacter colonies were tested biochemically by catalase and oxidase and subjected to Gram staining18.
PCR-based confirmation of C. jejuni: Genomic DNA were obtained using a conventional boiling method according to De Lamballerie et al.19. Campylobacter jejuni was distinguished from other Campylobacter spp. by the uniplex PCR-targeting mapA gene (Table 1).
| Table 1: | Oligonucleotide primers used in the study |
PCR reactions consisted of 12. 5 μL of 2×PCR master mix (Takara RR310A), 6 μL DNA template and 1 μL of each primer; the volume was completed to 25 μL with nuclease-free water. PCR conditions were followed according to the referenced study (Table 2).
Molecular characterization of cdtA and flaA genes: Campylobacter jejuni isolates were examined for the presence of cdtA and flaA genes (Table 1). The PCR reaction mixture for both genes consisted of 12.5 μL of 2×PCR master mix (Takara RR310A), 6 μL DNA template and 1 μL of 20 pmol of forward and reverse primer, the mixture was completed to 25 μL with DNA/RNA-free water (Table 2).
Poultry infections: Twenty one-day-old broiler chicks were used in this study. At one and 14 days of age, cloacal swabs were taken from all chickens and directly plated on mCCDA for Campylobacter determination to ensure absence of C. jejuni. At 14 days of age, each chicken was orally dosed with 1×108 C. jejuni in 0.5 mL of phosphate buffer saline23. Chicks were reared under strict biosecure conditions. At 7 days post-infection, the chicks were killed by cervical dislocation. One gram of cecal content was collected for C. jejuni analysis. Cecal contents were streaked on the surface of mCCDA and incubated under microaerobic condition at 42°C for 48 h24.
Histopathology: For histomorphological analysis, tissue samples were taken from the jejunum, liver and spleen and fixed in 4% buffered formalin for 48 h. The formalin fixed tissue was processed and stained using hematoxylin and eosin according to Lynch et al.25.
RESULTS
A total of 200 samples were collected from 100 chickens as follows: 50 samples from the liver, 50 samples from the spleen and 100 cecal swab samples. Each sample was tested for the presence of C. jejuni by isolation on mCCDA followed by the confirmatory PCR-targeting mapA gene (Fig. 1). Sixty eight C. jejuni isolates were recovered (18 from the liver samples, 12 from spleen samples and 38 from cecal swab samples), with an overall prevalence frequency of 34% (68/200).
Virulence genes were determined by uniplex PCR-targeting flaA and cdtA genes. The fla A gene was identified in all Campylobacter isolates used in this study, while cdtA was identified in only 62 (91.17%) Campylobacter isolates (Table 3, Fig. 2-3).
Campylobacter were successfully isolated from chicken ceca after infection in 2-week-old chicks. We identified several histopathological changes in the chicken gut from infection. There were alterations in the small intestinal architecture including, loss of tips, thickening and shortening of villi, inflammatory cell infiltration, sub-epithelial edema and edema and hemorrhage in serosal blood vessels (Fig. 4-6). However, no pathological changes were found in the liver and spleen.
| Table 2: | Cycling conditions of the primers during PCR |
| Fig. 1: | Agarose gel electrophoresis showing amplification of 589 bp fragment mapA gene |
| L: Ladder, Lane: 1, 2, 5, 6.9, 11, 12, 13, 15, 16, 17 and 18 positive for mapA gene, Neg: Negative control, Pos: Positive control | |
| Table 3: | Prevalence of Campylobacter jejuni in chicken samples |
| Fig. 2: | Agarose gel electrophoresis showing amplification of 855 bp fragment flaA primer |
| L: Ladder, Lane: 2, 3 and 4 positive for flaA gene, Neg: Negative control, Pos: Positive control | |
| Fig. 3: | Agarose gel electrophoresis showing amplification of 165 bp fragment of cdtA gene |
| L: Ladder, Lane: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 positive for cdt A gene, Neg: Negative control, Pos: Positive control | |
| Fig. 4: | Small intestine shows loss of tips of villi HE, ×: 100 arrows |
| Fig. 5: | Small intestine in broiler showing thickening and shortening of some villi HE, ×: 200 |
| Fig. 6: | Small intestine showing: Congestion and edema asterisk and hemorrhage in serosal blood vessels. HE, ×: 100 |
DISCUSSION
The presence of C. jejuni in internal organs indicates that Campylobacter can cross the intestinal barrier of animals and humans through bacterial translocation, which may occur via the lymphatic path or venous system11. In this study, the isolation rate of C. jejuni (34%) was relatively high, which indicates that this may be a significant avian pathogen; these results are consistent with previous studies26-29. Campylobacter jejuni was isolated from cloacal swabs, as well as the liver and spleen, which is also consistent with previous studies30-32. Campylobacter frequency may vary from one country to the other depending on many factors, such as hygienic measures and seasonal variations.
The flaA gene is involved in the invasion of the intestinal mucusa33. Therefore, its absence indicates severe reduction in motility and colonization of the human and chicken gut34. In this study, PCR showed that the flaA gene was present in all isolated C. jejuni strains, which is consistent with previous studies35-37.
Cytolethal distending toxin is conserved among Campylobacter strains and causes direct DNA damage, which leads to induction of DNA damage checkpoint pathways38,39. Three genes, (cdtA, cdtB and cdtC), are required to produce CDT and complete cellular toxicity40. In this study, 62 (91.17%) C. jejuni isolates carried the cdtA gene, which is in agreement with Bang et al.37, who found that the frequency of these genes exceeds 90% in Campylobacter isolates from different sources37. In addition, Hanning et al.36 reported that all Campylobacter isolates test were positive for the presence of cdtA and flaA36.
The role of CDT in pathogenesis is undistinguishable; however, there are many studies on its toxic effect on cultured mammalian cells16,41,42. According to a study conducted in Bangladesh by Talukder et al.43, CDT was identified as a virulence determinant which lead to increase fluid secretion in the intestine that causes diarrhea.
In the current study, experimental infection of commercial chickens with C. jejuni was associated with intestinal inflammation and pathological changes which is a harmful pathogen. Histopathological examination revealed many pathological changes including villi atrophy, which could explained by production of cytolethal distending toxin44. However, the role of other bacteria in the intestinal microbiota cannot be excluded45,46.
ACKNOWLEDGMENTS
The authors acknowledge the help of Dr. Mark Berrang, USDA Agricultural Research Service, U.S. National Poultry Research Center, Athens, GA 30605, USA for editing the manuscript.
SIGNIFICANCE STATEMENT
This study shows that Campylobacter infection is associated with intestinal inflammation and other pathological changes. This study revealed significant effects of C. jejuni infection on commercial chickens. These findings confirm that C. jejuni can have a significant effecton poultry health and welfare and is a harmful gut commensal microorganism. Thus, strict control measures are required to reduce its colonization.