Molecular Detection of Virulence-Associated Markers in Campylobacter coli and Campylobacter jejuni Isolates From Water, Cattle, and Chicken Faecal Samples From Kajiado County, Kenya

Campylobacter is a zoonotic foodborne pathogen that is often linked with gastroenteritis and other extraintestinal infections in humans. This study is aimed at determining the genetic determinants of virulence-encoding genes responsible for flagellin motility protein A (flaA), Campylobacter adhesion to fibronectin F (cadF), Campylobacter invasion antigen B (ciaB) and cytolethal distending toxin (cdt) A (cdtA) in Campylobacter species. A total of 29 Campylobacter coli isolates (16 from cattle, 9 from chicken, and 4 from water samples) and 74 Campylobacter jejuni isolates (38 from cattle, 30 from chicken, and 6 from water samples) described in an earlier study in Kajiado County, Kenya, were examined for the occurrence of virulence-associated genes using polymerase chain reaction (PCR) and amplicon sequencing. The correlations among virulence genes were analyzed using Pearson's correlation coefficient (R) method. Among the 103 Campylobacter strains screened, 89 were found to harbour a single or multiple virulence gene(s), giving an overall prevalence of 86.4%. C. jejuni strains had the highest prevalence of multivirulence at 64.9% (48/74), compared to C. coli (58.6%, 17/29). The ciaB and flaA genes were the most common virulence genes detected in C. jejuni (81.1% [60/74] and 62.2% [46/74], respectively) and in C. coli (each at 62.1%; 18/29). Campylobacter isolates from chicken harboured the most virulence-encoding genes. C. jejuni strains from chicken and cattle harboured the highest proportions of the cdtA and ciaB genes, respectively. All the C. coli strains from water samples harboured the cadF and flaA genes. The results obtained further revealed a significant positive correlation between cadF and flaA (R = 0.733). C. jejuni and C. coli strains from cattle, chicken, and water harbour virulence markers responsible for motility/colonization, invasion, adherence, and toxin production, evoking their important role in campylobacteriosis development among humans and livestock. The identification of cattle, chicken, and water samples as reservoirs of virulent Campylobacter spp. highlights the possible risk to human health. These data on some virulence genes of Campylobacter will assist food safety and public health officials in formulating policy statements.


Introduction
Campylobacter spp.particularly Campylobacter coli and Campylobacter jejuni are major causative agents of bacterial gastroenteritis infections among humans in developing and developed countries [1].Livestock including chicken play a significant role in Campylobacter infections in humans.The role of cattle and chicken as sources of Campylobacter infections in humans is related to (1) contaminated foods such as raw milk, beef, and poultry meat; (2) environmental and water pollution; and (3) direct spread to humans from infected animals.Of these, uncooked or undercooked chicken meat is the most likely source of Campylobacter spp.[2], often linked with sudden and unpredictable cases of human campylobacteriosis.There are also bovinerelated epidemics where unpasteurized milk and its byproducts are the second most prevalent sources of infection [3].However, other reservoirs of infection, such as companion animals, raw milk, and contaminated water, have been noted [4].Direct contamination of milk might occur via either faeces or as a result of udder infection caused by these pathogens [5,6].
Campylobacter spp.not only impact human health but also cause significant infections in livestock.Campylobacter infections in humans and livestock present a variable degree of virulence: ranging from asymptomatic carriage in poultry to gastroenteritis and/or watery diarrhea and sometimes extraintestinal infections (respiratory failure and severe neurological dysfunction) in humans, to mastitis, enteritis, and abortion in cattle [7].However, the precise role of Campylobacter isolates in the causation of all these clinical syndromes is unclear, and additional research is consequently necessary [8].The pathogenesis of campylobacteriosis is multifaceted and still not well understood [9].Exploring the molecular basis of the virulence markers linked with the pathogenicity of Campylobacter spp. is essential for controlling diseases and/or clinical manifestations caused by this bacterium [10].However, polymerase chain reaction (PCR) virulotyping of Campylobacter spp.has been widely investigated in other countries [11].Consequently, some studies have explored the potential virulence and/or survival factors necessary for the pathogenicity of Campylobacter spp.[12]; the response to stress, flagellum-facilitated motility, adhesion and binding, invasion and adherence to epithelial cells of the intestines, chemotaxis, ability to produce toxins, and ability to overcome host defense cells are now known to be involved in pathological processes and/or disease development in Campylobacter spp.[11][12][13].However, it is not clear whether specific disease syndromes correlate with a particular virulence-encoding gene.Nevertheless, virulence attributes are believed to contribute to the organism's pathogenicity and provide the ability to adhere to receptor cells on the host during disease pathogenesis, hence helping to modulate the clinical manifestations of the disease [14].
A number of genes and gene products have been documented to play vital roles during disease development.Campylobacter invasion antigen B (ciaB) is an important heat shock protein-encoding gene [15] that is necessary for invasion of the epithelium and subsequent establishment in avian intestines [16].However, effective invasion and establishment of Campylobacter spp.into host epithelial cells depend on its adhesion to fibronectin F, which is encrypted by the Campylobacter adhesion to fibronectin F (cadF) gene and is responsible for the binding of this bacterium to the intercellular matrix of epithelial cells in the intestines during the disease development process [13].Cytolethal distending toxins (cdts) are enciphered by a family of genes, namely, cdt A (cdtA), cdtB, and cdtC, and are essential for the cytotoxicity and destruction of intestinal absorptive cells in the host [17].The flagellum motility protein is encrypted by the flagellin motility protein A (flaA) gene and is implicated in motility, establishment, autoagglutination, and biofilm development, thus contributing to the Campylobacter infection process in a susceptible host [18].
Campylobacter strains originating from different sources, including livestock and their environment, may be transferred to humans, causing gastroenteritis, among other clinical syndromes.Therefore, it is imperative to establish whether Campylobacter strains recovered from these sources possess virulence properties.Furthermore, virutyping Campylobacter isolates could be an important step in understanding the progression of associated infections in humans.Therefore, surveillance of virulence genetic determinants in Campylobacter spp. is exceedingly applicable to consumer health.In anticipation of this context, the objective of the present study was to establish the occurrence of virulence-encoding genes responsible for flaA, cadF, ciaB, and cdtA in Campylobacter species recovered from cattle, chicken, and water samples from Kajiado County, Kenya.

Materials and Methods
2.1.Ethical Approval.The study was reviewed and authorized by the Biosafety, Animal Use and Ethics Committee, Faculty of Veterinary Medicine, University of Nairobi (FVM/BAUEC/2020/274).

Study
Area, Design, Source, and Culture Conditions of Campylobacter Strains.Bacterial strains utilized in the virulotyping assays were obtained from a cross-sectional investigation on seasonal occurrence on thermotolerant Campylobacter spp.isolated from cattle rectal swabs, chicken cloacal swabs, and water samples from cattle drinkers and other designated cattle's waterpoints, conducted in Kajiado County, southwestern Kenya.The study site locations, procedure of sampling, isolation, and identification of the isolates were as described previously [19,20].Briefly, a prevalence survey of 55 mixed livestock farms (primarily keeping cattle and chicken) was carried out in three subcounties in Kajiado County (Ongata Rongai and Ngong in Kajiado North subcounty; Kiserian in Kajiado West subcounty; and Kitengela, Isinya, and Mashuru in Kajiado East subcounty) (Figure 1).
The county has well-established smallholder mixed livestock (cattle, poultry, sheep, and goats among others) production systems.The mixed farms were visited once, and a single faecal swab (from cattle and chicken) and water samples were collected either during the rainy or dry season, 2 BioMed Research International which spans the period between October 2020 and May 2022.
Isolation of Campylobacter species from water samples was via aseptic filtration through 0.45 μm filter paper.Thereafter, the filter paper and faecal swabs were processed by initial selective pre-enrichment in Bolton broth (Oxoid, UK), followed by routine isolation and culture for 48 h at 42 °C onto modified charcoal-cefoperazone-deoxycholate agar (mCCDA)/Campylobacter agar plates (Oxoid, UK) under microaerobic conditions.Pure cultures of bacteria were obtained by aseptically streaking putative colonies on freshly prepared blood agar plates with selective supplement.Putative Campylobacter isolates were identified via both conventional biochemical methods and singleplex-PCR.Prior to the PCR assay, culture-based confirmed Campylobacter isolates were subjected to DNA extraction via the boiling method and then frozen at −80 °C.Singleplex-PCR identification revealed that 55.6% (90/162) were C. jejuni and 17.9% (29/162) were C. coli.A total of 103 PCR-confirmed Campylobacter isolates including 29 C. coli (16 from cattle, 9 from chicken, and 4 from water samples) and 74 C. jejuni (38 from cattle, 30 from chicken, and 6 from water samples) isolates were selected for the current virulence study.Subsequently, the previously cryopreserved genomic DNA from these Campylobacter isolates was utilized for the virutyping assays.

Molecular Detection of Virulence
Genes.The cryopreserved genomic DNA was removed from the freezer and allowed to thaw at room temperature (24 °C-26 °C), prior to being subjected to virutyping assays.PCR was utilized to screen virulence-associated genes including flaA, ciaB, cdtA, and cadF.The oligonucleotide primers used were designed based on gene sequence data from previously published reports (Table 1).The PCR primers used were sourced from Inqaba Biotechnologies (Pretoria, South Africa).The primer sequences were subjected to Basic Local Alignment Search Tool (BLAST) searches against the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov) to assess their specificity.
The cryopreserved genomic DNA samples were thawed and subsequently amplified using primers specific for each of the virulence markers in a Bio-Rad thermal cycler.The final PCR mixture included 12.5 μL of OneTaq® 2x PCR Master Mix (New England Biolabs), 0.2 μL of each primer pair (cadF-R/F, cdtA-R/F, flaA-R/F, and ciaB-R/F), and 5 μL of sample DNA.The final volume was topped up to 25 μL using molecular-grade water (BioConcept®, Switzerland).The following optimized multiplex-PCR (mPCR) thermal cycling conditions for cdtA, flaA, and ciaB genes were used: 95 °C × 5 min (initial denaturation); 30 cycles 94 °C × 1 min, 57 °C × 1 min (annealing); 72 °C × 1 min; 72 °C × 5 min (terminal extension).Singleplex amplification conditions for cadF were as described for mPCR, except that the annealing temperature was 48 °C for 1 min.C. jejuni subsp.jejuni ATCC 33560 and molecular-grade water were used as positive and negative controls in each PCR run, respectively.The amplified PCR products were separated 3 BioMed Research International via electrophoresis on a 1.5% agarose gel, stained with ethidium bromide in 1x Tris-Borate-EDTA (TBE) buffer (Cleaver Scientific Ltd, UK), and visualized via ultraviolet light transillumination using a gel document system (Gel-Max® 125 UVP imager, Cambridge, UK).The sizes of the PCR amplicons were compared to that of the 100 bp DNA ladder.
2.4.Virulence-Encoding Amplicon Sequencing.Representative amplicons encoding virulence genes generated with each primer were purified using the QIAquick PCR Purification Kit (Qiagen) and then shipped to Inqaba Biotechnologies laboratories, Pretoria, South Africa, for sequencing using both forward and reverse primers.The raw sequences were edited, aligned, and assembled into a complementary sequence using BioEdit software.The sequences were blasted against the NCBI GenBank database for the best matches.The sequences were subsequently submitted to GenBank to obtain accession numbers.
2.5.Data Handling and Analysis.Data on detection of virulence-encoding gene was entered into and stored in Microsoft Excel and subsequently authenticated before descriptive and inferential statistical analyses were performed via EPI INFO software (https://www.cdc.gov/epiinfo/).Pearson's correlation coefficient (R) was used to establish associations among the virulence genes to determine whether the presence of any of the given genes was interconnected with the other.Chi-square and Fisher's exact tests were employed to examine whether the detected virulence markers were influenced by the source of the isolates (cattle, chickens, or water).A p value < 0 05 was considered to indicate statistical significance.

Detection of Virulence Genes Among Campylobacter
Isolates.Conventional PCR analysis revealed Campylobacter isolates harbouring genetic determinants responsible for various virulence factors.As shown in Figure 2, PCR bands corresponding to 370 bp for the cdtA gene (Figure 2(a)) and 400 bp for cadF (Figure 2(b)) were detected.The other virulence genes yielded specific bands corresponding to 527 bp for ciaB (Figure 2(c)) and 855 bp for flaA (Figure 2(d)).
The frequency of detection of virulence-encoding genes among Campylobacter strains irrespective of the source/ sample type in this study is presented in Figure 3 4.There were significant differences noted between the proportions of virulenceencoding genes found in cattle, chicken, and water (p value < 0 05).Chicken-Campylobacter strains harboured the majority of virulence-encoding genes.The results further showed variability in the proportions of virulence genes among Campylobacter species from the diverse sources (Figure 5).The highest detection rate of cdtA was detected among C. jejuni isolates from chicken (56.7%; 17/30), 34.2% (13/38) and 33.3% (2/6) of which were from cattle and from water, respectively.Similarly, 33.3% (3/9) of the C. coli isolates from chicken harboured cdtA, 25% (4/16) from cattle, and 25% (1/4) from water, as shown in Figure 5(a).All the four C. coli isolates from water (100%,

Pearson's Correlations for Virulence-Associated Genes
Detected in Campylobacter Species.Statistically significant positive correlations were noted between several virulence genes assayed in this study (p < 0 05) (Table 2).The ciaB gene was the only gene that was not significantly correlated with the cdtA gene (p > 0 05).The presence of the cadF gene was strongly correlated with the presence of the flaA gene (R = 0 733).The occurrence of cdtA (one of the tripartite cdts), which causes unwinding of DNA strands, was moderately correlated with the presence of both the cadF (R = 0 645) and flaA (R = 0 544) genes (p < 0 05).

Nucleotide Sequence Accession
Numbers.Some nucleotide sequences from this study were submitted to the Gen-Bank and assigned accession numbers as follows: cadF gene (OR876350, OR876351, and OR876352) and flaA gene (OR876353, OR876354) in the NCBI databases available at https://www.ncbi.nlm.nih.gov/nucleotide.

Discussion
Virulence-encoding genes are responsible for Campylobacter's pathogenicity; therefore, virulence-associated factors in livestock (cattle and chicken) and nonlivestock/environmental (water) reservoirs warrant studies for the sake of human safety.There are limited studies that have investigated virulence-related genes in Campylobacter strains of environmental origin including water, as most of them have focused on the occurrence of virulence markers in Campylobacter strains in humans and domestic animals, particularly poultry.This study subsequently investigated genes encoding virulence markers, including cdtA, flaA, ciaB, and cadF, in cattle, chicken, and water samples.In the current study, both C. jejuni and C. coli isolates were found to harbour multiple virulence genes at significantly higher frequencies.This finding corroborates findings in another study by Bunduruș et al. [25].The presence of several virulence factors in a single strain increases the invasiveness of this bacterium.Consequently, the findings also imply that both C. jejuni and C. coli from the three sample types could be equally culpable for most of the clinical syndromes.
Overall, the ciaB gene, which is responsible for attacking host epithelial cells, was the most common virulenceencoding gene detected in this study, occurring in 79.5%, 77.8%, and 50% of the chicken, cattle, and water isolates, respectively.Thus, the chicken isolates presented the highest proportion of the ciaB gene.The detection rates of the ciaB gene in C. jejuni (83.3%) and C. coli (66.7%) isolates from chicken were comparable with the findings of previous stud-ies [26].However, the percentage of the ciaB gene among chicken isolates stated in this study was greater than that reported in other studies: 47% and 10% in C. jejuni and C. coli isolates, respectively [27], and 23.1% among Campylobacter isolates [28].The ciaB gene was identified in 56.3% and 86.8% of the C. coli and C. jejuni strains, respectively, from cattle, which is akin to the findings of Raeisi et al. [29].The current study further showed a high proportion (75% of C. jejuni and 33.3% of C. coli) of the ciaB gene in water isolates, and the results are comparable with the results of the study by Chukwu et al. [30].On the contrary, Igwaran and Okoh [31] reported no prevalence of the same gene in Campylobacter isolates from water samples from both ponds/dams and rivers.The ciaB gene is necessary for the early phases of Campylobacter establishment [18]; therefore, the dominance of the latter in cattle, chicken, and water samples means that the recovered strains were able to overcome adverse intestinal conditions and instigate disease processes [18].Additionally, this gene had a significantly low positive correlation of 19.7% and 21.2% with the cadF and flaA genes, respectively (p < 0 05).This finding is significant because the first stage in the pathogenesis of invasive versus toxigenic pathogens involves attachment to host cells.Thus, the ciaB gene is essential for pathogenic Campylobacter strains, which, despite lacking other virulence genes, could result in infections.
The flagellin protein FlaA, a major protein encoding the flaA gene, was the second most commonly detected putative virulence gene.The flaA gene is essential for bacterial motility and establishment in epithelial cells of the ileum.6 BioMed Research International Furthermore, the flaA gene also accounts for Campylobacter attachment, attack, and establishment in host epithelial cells, thereby halting the immune response [26].The presence of either the flaA or cadF gene results in attachment and hence increased likelihood of successful disease development.Studies have reported the presence of the flaA gene in C. jejuni and C. coli strains recovered from chicken [26,32], cattle [26], and water samples [32]; the findings of this article as well conform with these studies.However, a higher incidence of up to 100% has been reported for Campylobacter species from diverse sources [33,34], with higher detection of the flaA gene being associated with high conservation of the FlaA locus among Campylobacter strains.
Based on the findings of this study, 61.5%, 60%, and 38.9% of Campylobacter strains from chicken, water, and cattle samples, respectively, harboured the cadF gene.In chicken, 66.7% of C. jejuni and 44.4% of C. coli strains possessed the cadF gene, which is concordant with the findings of Ngobese, Zishiri, and El Zowalaty [26].C. coli-cadF genepossessing isolates of bovine origin were also reported by Wieczorek and Osek [33]; however, a much greater frequency (90%-100%) of the cadF gene in both C. jejuni and C. coli isolates has also been reported [34,35].All the  7 BioMed Research International water-derived C. coli isolates harboured cadF markers.Similarly, a greater proportion of the cadF gene (100%) was detected in C. coli strains isolated from water samples from rivers, freshwater beaches, lakes, and ponds in northern Poland [32].
Campylobacter spp.are known to produce cdts (encrypted by cdtA, cdtB, and cdtC), which cause DNA destruction, chromatin disintegration, cytoplasmic distension, and halt mitotic cell division, resulting in cumulative cellular distension and eventually pathogen-induced host necrobiosis [26].In this study, regardless of Campylobacter spp., 51.3% of the cdtA gene was found in chicken isolates, 33.3% in C. coli, and 34.2% in C. jejuni.More or less similar findings were reported by Ramatla et al. [28], who reported that 26.9% of C. jejuni isolates from broilers harboured the cdtA gene.Ngobese, Zishiri, and El Zowalaty [26] also described a high assemblage rate of the cdtA gene in chicken samples, but at a much greater percentage (96% of C. jejuni and 83% of C. coli).Additionally, the C. jejuni isolates from cattle in the present study harboured the most cdtA genes (56.7%).Studies have reported discrepant detection rates-37% for C. jejuni, 50% for C. coli [26], and 100% in both C. jejuni and C. coli [33], for this gene among Campylobacter strains of cattle origin.Limited data exist on the occurrence of virulence markers (including cdtA) among Campylobacter strains from the environmental water.These results can subsequently be compared with findings on other cdt-encoding genes (cdtB or cdtC), where studies have detected varying incidence of the cdtB gene in Campylobacter strains found in water samples [31,32].The presence of cytotoxic genes (cdtA among others) among livestock and environmental sources highlights a food safety alarm as well as a public health warning.
Consistent with global observations, this study identified the incidence of virulence-encoding genes in Campylobacter, more specifically motility, CDT production, and ability to invade and adhere to epithelial cells, and these data are in accordance with studies recently conducted in developed European nations [36][37][38][39][40].These studies have equally emphasized that (1) these genes mainly encipher for factors essential in the early stages of infection and (2) poultry and cattle among other animals are the primary sources of pathogenic Campylobacter strains to humans.The findings of the current study, however, revealed relatively greater proportions of virulence-associated genes than did other studies.This study evokes that the observed discrepancies may be due to primer specificity, PCR protocols, climate and/or environmental conditions, geographical factors and seasonality, freezing, and thawing.Additionally, these virulenceencoding genes are carried on plasmids, which may influence their occurrence in different strains [41].Additionally, the virulence markers described in this study have also been documented in Campylobacter species of human origin [27,41,42], emphasizing the probable role of these strains in causing Campylobacter infections in humans.However, it is crucial to highlight that the occurrence of virulence markers is merely indicative/suggestive and unpredictable of how deadly a Campylobacter strain might be.To rule out this possibility, a comparative analysis of patient demographics and the interaction between virulence and presenting symptoms are needed to prove disease causation, as the severity of a disease depends on the virulence of strain involved and host immunity [43].

Conclusions
C. jejuni and C. coli strains from bovine, chicken, and water harbour multiple virulence markers responsible for motility/ colonization (flaA), invasiveness (ciaB), adherence (cadF), and toxin production (cdtA), evoking their important role in campylobacteriosis development among humans and livestock.The virulence-encoding markers were more prevalent in the chicken samples than in the other sample types.Additionally, regardless of the sample type, C. jejuni strains exhibited the highest detection rate of multiple virulence markers in this study.Additional studies involving fullgenomic sequencing to evaluate other genetic markers for virulence would assist in elaborating the possible role of chicken, cattle, and environmental waters as vehicles in the epidemiology of campylobacteriosis disease in humans.

Data Availability Statement
The raw datasets used in this study are available from the corresponding author (wanjadanie@gmail.com)upon reasonable request.

Figure 1 :
Figure 1: A map showing the location of Kajiado County, Kenya (black stars show study locations).

3. 3 .
Prevalence of Virulence-Associated Genes in C. jejuni and C. coli Isolated From Chicken, Cattle, and Water Samples.

Figure 3 :
Figure 3: Percentages of virulence-encoding genes in C. coli and C. jejuni strains from all the sample types.The data are presented as the percentage fraction ± standard deviation (C. coli [n = 29], C. jejuni [n = 74]).

Table 1 :
Primers used for virulence gene typing in this study.

Table 2 :
Assessment of Pearson's correlations for virulence-encoding genes in relation to the other genes in a single sample type in Campylobacter strains.